Lafora&#39;s disease gene

ABSTRACT

A novel gene (EPM2A) that is deleted or mutated in people with Lafora&#39;s disease is described. The EPM2A gene encodes a protein having an active catalytic site of a protein tyrosine phosphatase. Many different sequence mutations as well as several microdeletions in EPM2A have been found that co-segregate with Lafora&#39;s disease.

FIELD OF THE INVENTION

[0001] The invention relates to a novel gene, EPM2A, that is involved inLafora's disease; the protein, Laforin, encoded by the gene; and methodsof diagnosing and treating Lafora's disease.

BACKGROUND OF THE INVENTION

[0002] The epilepsies constitute one of the most common neurologicaldisorders affecting 40 million people worldwide (1). Within the spectrumof epileptic syndromes is a group of heterogeneous inherited disordersnamed the Progressive Myoclonus Epilepsies (PME) in which progressiveneurological decline and worsening primarily myoclonic seizures followan initial period of normal development (2,3,4). Lafora's disease (LD)is an autosomal recessive and genetically heterogeneous form ofProgressive Myoclonus Epilepsy characterized by polyglucosan inclusionsseizures and cumulative neurological deterioration. The onset occursduring late childhood and usually results in death within a decade offirst symptoms. With few exceptions, patients with LD follow ahomogeneous clinical course (4) despite the existence of genetic locusheterogeneity (5). Biopsy (or autopsy) of various tissues includingbrain, liver, muscle, and skin reveals characteristic periodicacid-Schiff positive polyglucosan inclusions (Lafora bodies) (6-9).Substantial biochemical and histological studies of these bodies suggestLD is a generalized storage disease (8,10,11), but the presumedenzymatic defect remains unknown.

[0003] Linkage analysis and homozygosity mapping initially localized aLafora's disease locus (EPM2A) to a region at chromosome 6q23-q25bounded by the genetic markers D6S1003 and D6S311 (12,13). However,there is a need in the art to more clearly define the region(s) mutatedin Lafora's disease to allow for the development of accurate diagnosticassays for Lafora's disease. More specifically, there is a need tosequence the gene associated with Lafora's Disease and to identifymutations and/or deletions in the gene that are causative of Lafora'sDisease.

SUMMARY OF THE INVENTION

[0004] The present inventors have identified a novel gene, EPM2A, thatis deleted or mutated in people with Lafora's disease. Using apositional cloning approach the inventors have identified at chromosome6q24 the EPM2A gene that encodes a protein with consensus amino acidsequence indicative of a tyrosine phosphatase. Accordingly, the presentinvention provides an isolated nucleic acid molecule containing asequence encoding an active catalytic site of a protein tyrosinephosphatase which is associated with Lafora's disease.

[0005] In one embodiment of the invention, an isolated nucleic acidmolecule is provided having a sequence as shown in SEQ.ID.NO.:1 or FIG.13.

[0006] Preferably, the purified and isolated nucleic acid moleculecomprises:

[0007] (a) a nucleic acid sequence as shown in SEQ.ID.NO.:1 and FIG. 13,wherein T can also be U;

[0008] (b) nucleic acid sequences complementary to (a);

[0009] (c) nucleic acid sequences which are homologous to (a) or (b);

[0010] (d) a fragment of (a) to (c) that is at least 15 bases,preferably 20 to 30 bases, and which will hybridize to (a) to (d) understringent hybridization conditions; or

[0011] (e) a nucleic acid molecule differing from any of the nucleicacids of (a) to (c) in codon sequences due to the degeneracy of thegenetic code.

[0012] Fourteen different mutations in EPM2A in 24 families have beenfound that co-segregate with Lafora's disease. These alterations wouldbe predicted to abolish or cause deleterious effects on the proteinproduct, Laforin, resulting in the primary defect in a large portion ofpatients with the disease. Accordingly, the present invention provides amethod of detecting Lafora's disease comprising detecting a mutation ordeletion in the EPM2A gene in a sample from a mammal. A mutation can bedetected by sequencing the EPM2A gene, in particular in the region inthe gene between markers D6S1003 and D6S1042, in a patient and comparingthe sequence to the wild type EPM2A sequence shown in FIG. 13 todetermine if a mutation or deletion is present. A mutation or deletioncan also be detected by assaying for the protein product encoded byEPM2A, Laforin.

[0013] Other features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples while indicating preferred embodiments of the invention aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The invention will now be described in relation to the drawingsin which:

[0015]FIG. 1 is a physical map of the Lafora's disease critical region.

[0016]FIG. 2A shows a refined mapping of the Lafora disease gene forLafora family LD39.

[0017]FIG. 2B is for Lafora family LD-L4.

[0018]FIG. 3 shows overlapping cDNA clones aligned with genomic DNAsegments.

[0019]FIG. 4A is the nucleotide sequence and predicted amino acidsequence of EPM2A (incomplete).

[0020]FIG. 4B is an amino acid sequence of the carboxy terminus oftranscript A compared to transcript B.

[0021]FIG. 4C shows the PTP action sites of EPM2A, MTMI (Swiss prot.C13496), PTEN (Swiss prot, 000633, PTP 1B (Swiss prot APT P61F (GenBankLI4849) and viral PTP (Swiss prot Af 003534).

[0022]FIG. 5 is a Northern blot showing RNA expression pattern of EPM2A.

[0023]FIG. 6A shows representative mutations found in Lafora's familyLD-16.

[0024]FIG. 6B shows Lafora's family LD-33.

[0025]FIG. 7 is a nucleotide sequence of transcript A cDNA of the EPM2Agene (SEQ.ID.NO.:3).

[0026]FIG. 8 is the predicted amino acid sequence of transcript A(SEQ.ID.NO.:4).

[0027]FIG. 9 is a nucleotide sequence of transcript B cDNA of the EPM2Agene (SEQ.ID.NO.: 5).

[0028]FIG. 10 is the predicted amino acid sequence of transcript B(SEQ.ID.NO.: 6).

[0029]FIG. 11 is a refined map of the deletion breakpoints in familiesLD-L4, LD9 and LD1.

[0030]FIG. 12A is a restriction map of PCR products with primersH1F/PTPR.

[0031]FIG. 12B is the HaeIII and PstI digestion of the H1F/PTPR PCRproduct.

[0032]FIG. 13 is the complete nucleic acid sequence of EPM2A. This isalso shown in SEQ.ID.NO.:1.

[0033]FIG. 14 is the complete amino acid sequence of EPM2A. This is alsoshown in SEQ.ID.NO.:2.

DETAILED DESCRIPTION OF THE INVENTION

[0034] The present inventors constructed a high resolution physical mapacross the EPM2A gene to provide additional genetic and physical mappingreagents for refined localization of the disease gene. It was determinedthat the previously established critical region encompassedapproximately 1.2 Mb of DNA. The map allowed the positioning of thelocation of 7 genetic markers, the metabotropic glutamate receptor 1(GRM1) gene, and 6 expressed sequence tags (EST) clusters (tentativelynamed LDCR1-LDCR6), within the interval (FIG. 1). The genetic markerswere then used to test for regions of homozygosity in each of the 30families with Lafora's disease that appeared genotypically to arise dueto mutations in a gene at 6q23-q25. In a single family (LD39), anextended chain of homozygous markers within the previously establishedcritical region allowed the inventors to, tentatively, redefine thetelomeric boundary at D6S1042 (FIG. 2A). Simultaneously, a homozygousdeletion of marker D6S1703 in the affected of a consanguineous family(LD-L4) (FIG. 2B) was detected. This observation confirmed the newlydefined critical region to that 600 kb of DNA between D6S1003 andD6S1042, but more importantly, pinpointed the site of the disease genewithin this region.

[0035] I. Nucleic Acid Molecules of the Invention

[0036] As hereinbefore mentioned, the present invention relates toisolated nucleic acid molecules that are involved in Lafora's disease.The term “isolated” refers to a nucleic acid substantially free ofcellular material or culture medium when produced by recombinant DNAtechniques, or chemical precursors, or other chemicals when chemicallysynthesized. The term “nucleic acid” is intended to include DNA and RNAand can be either double stranded or single stranded.

[0037] Broadly stated, the present invention provides an isolatednucleic acid molecule containing a sequence encoding an active catalyticsite of a protein tyrosine phosphatase which is associated with Lafora'sdisease. The isolated nucleic acid molecule is preferably the EPM2A geneassociated with Lafora's disease. In an embodiment of the invention, theisolated nucleic acid molecule has a sequence as shown in SEQ.ID.NO.:1and FIG. 13.

[0038] Preferably, the purified and isolated nucleic acid moleculecomprises

[0039] (a) a nucleic acid sequence as shown in SEQ.ID.NO.:1 and FIG. 13,wherein T can also be U;

[0040] (b) nucleic acid sequences complementary to (a);

[0041] (c) nucleic acid sequences which are homologous to (a) or (b);

[0042] (d) a fragment of (a) to (c) that is at least 15 bases,preferably 20 to 30 bases, and which will hybridize to (a) to (d) understringent hybridization conditions; or

[0043] (e) a nucleic acid molecule differing from any of the nucleicacids of (a) to (c) in codon sequences due to the degeneracy of thegenetic code.

[0044] The inventors have also isolated alternate forms of EPM2A whichare generally referred to as transcript A and transcript B, herein. Thenucleic acid sequence of transcript A is shown in SEQ.ID.NO.:3 and FIG.7. The nucleic acid sequence of transcript B is shown in SEQ.ID.NO.:5and FIG. 9. The amino acid sequence encoded by transcript A is shown inSEQ.ID.NO.:4 and FIG. 8. The amino acid sequence encoded by transcript Bis shown in SEQ.ID.NO.:6 and FIG. 10.

[0045] The nucleic acid sequences shown in SEQ.ID.NOS.: 1, 3 and 5 (orFIGS. 13, 7 and 9, respectively) can be collectively referred to hereinas “the nucleic acid molecules of the invention”. The amino acidsequences shown in SEQ.ID.NOS.: 2, 4 and 6 (or FIGS. 4A, 8 and 10,respectively) may be collectively referred to herein as the “proteins ofthe invention”.

[0046] It will be appreciated that the invention includes nucleic acidmolecules encoding truncations of the proteins of the invention, andanalogs and homologs of the proteins of the invention and truncationsthereof, as described below. It will further be appreciated that variantforms of the nucleic acid molecules of the invention which arise byalternative splicing of an mRNA corresponding to a cDNA of the inventionare encompassed by the invention.

[0047] Further, it will be appreciated that the invention includesnucleic acid molecules comprising nucleic acid sequences havingsubstantial sequence homology with the nucleic acid sequences of theinvention and fragments thereof. The term “sequences having substantialsequence homology” means those nucleic acid sequences which have slightor inconsequential sequence variations from these sequences, i.e. thesequences function in substantially the same manner to producefunctionally equivalent proteins. The variations may be attributable tolocal mutations or structural modifications.

[0048] Generally, nucleic acid sequences having substantial homologyinclude nucleic acid sequences having at least 70%, preferably 80-90%identity with the nucleic acid sequences of the invention.

[0049] Another aspect of the invention provides a nucleic acid molecule,and fragments thereof having at least 15 bases, which hybridizes to thenucleic acid molecules of the invention under hybridization conditions,preferably stringent hybridization conditions. Appropriate stringencyconditions which promote DNA hybridization are known to those skilled inthe art, or may be found in Current Protocols in Molecular Biology, JohnWiley & Sons, N.Y. (1989), 6.3.1-6.3.6. For example, the following maybe employed: 6.0× sodium chloride/sodium citrate (SSC) at about 45° C.,followed by a wash of 2.0×SSC at 50° C. The stringency may be selectedbased on the conditions used in the wash step. For example, the saltconcentration in the wash step can be selected from a high stringency ofabout 0.2×SSC at 50° C. In addition, the temperature in the wash stepcan be at high stringency conditions, at about 65° C.

[0050] Isolated and purified nucleic acid molecules having sequenceswhich differ from the nucleic acid sequence shown in SEQ.ID.NO.:1 orSEQ.ID.NO.:3 or SEQ.ID.NO.:5 due to degeneracy in the genetic code arealso within the scope of the invention.

[0051] Nucleic acid molecules from the EPM2A gene can be isolated bypreparing a labelled nucleic acid probe based on all or part of thenucleic acid sequences as shown in SEQ.ID.NO.:1 and FIG. 13, and usingthis labelled nucleic acid probe to screen an appropriate DNA library(e.g. a cDNA or genomic DNA library). Nucleic acids isolated byscreening of a cDNA or genomic DNA library can be sequenced by standardtechniques.

[0052] Nucleic acid molecules of the invention can also be isolated byselectively amplifying a nucleic acid using the polymerase chainreaction (PCR) methods and cDNA or genomic DNA. It is possible to designsynthetic oligonucleotide primers from the nucleic acid molecules asshown in SEQ.ID.NO.:1 and FIG. 13, for use in PCR. A nucleic acid can beamplified from cDNA or genomic DNA using these oligonucleotide primersand standard PCR amplification techniques. The nucleic acid so amplifiedcan be cloned into an appropriate vector and characterized by DNAsequence analysis. It will be appreciated that cDNA may be prepared frommRNA, by isolating total cellular mRNA by a variety of techniques, forexample, by using the guanidinium-thiocyanate extraction procedure ofChirgwin et al., Biochemistry, 18, 5294-5299 (1979). cDNA is thensynthesized from the mRNA using reverse transcriptase (for example,Moloney MLV reverse transcriptase available from Gibco/BRL, Bethesda,Md., or AMV reverse transcriptase available from Seikagaku America,Inc., St. Petersburg, Fla.).

[0053] An isolated nucleic acid molecule of the invention which is RNAcan be isolated by cloning a cDNA encoding a novel protein of theinvention into an appropriate vector which allows for transcription ofthe cDNA to produce an RNA molecule which encodes the Laforin protein.For example, a cDNA can be cloned downstream of a bacteriophagepromoter, (e.g. a T7 promoter) in a vector, cDNA can be transcribed invitro with T7 polymerase, and the resultant RNA can be isolated bystandard techniques.

[0054] A nucleic acid molecule of the invention may also be chemicallysynthesized using standard techniques. Various methods of chemicallysynthesizing polydeoxynucleotides are known, including solid-phasesynthesis which, like peptide synthesis, has been fully automated incommercially available DNA synthesizers (See e.g., Itakura et al. U.S.Pat. No. 4,598,049; Caruthers et al. U.S. Pat. No. 4,458,066; andItakura U.S. Pat. Nos. 4,401,796 and 4,373,071).

[0055] The initiation codon and untranslated sequences of the nucleicacid molecules of the invention may be determined using currentlyavailable computer software designed for the purpose, such as PC/Gene(IntelliGenetics Inc., Calif.). Regulatory elements can be identifiedusing conventional techniques. The function of the elements can beconfirmed by using these elements to express a reporter gene which isoperatively linked to the elements. These constructs may be introducedinto cultured cells using standard procedures. In addition toidentifying regulatory elements in DNA, such constructs may also be usedto identify proteins interacting with the elements, using techniquesknown in the art.

[0056] The sequence of a nucleic acid molecule of the invention may beinverted relative to its normal presentation for transcription toproduce an antisense nucleic acid molecule. Preferably, an antisensesequence is constructed by inverting a region preceding the initiationcodon or an unconserved region. In particular, the nucleic acidsequences contained in the nucleic acid molecules of the invention or afragment thereof, preferably a nucleic acid sequence shown inSEQ.ID.NO.:1, SEQ.ID.NO.:3 or SEQ.ID.NO.:5 may be inverted relative toits normal presentation for transcription to produce antisense nucleicacid molecules.

[0057] The antisense nucleic acid molecules of the invention or afragment thereof, may be chemically synthesized using naturallyoccurring nucleotides or variously modified nucleotides designed toincrease the biological stability of the molecules or to increase thephysical stability of the duplex formed with mRNA or the native genee.g. phosphorothioate derivatives and acridine substituted nucleotides.The antisense sequences may be produced biologically using an expressionvector introduced into cells in the form of a recombinant plasmid,phagemid or attenuated virus in which antisense sequences are producedunder the control of a high efficiency regulatory region, the activityof which may be determined by the cell type into which the vector isintroduced.

[0058] The invention also provides nucleic acids encoding fusionproteins comprising a novel protein of the invention and a selectedprotein, or a selectable marker protein (see below).

[0059] II. Novel Proteins of the Invention

[0060] The invention further includes an isolated protein encoded by thenucleic acid molecules of the invention. Within the context of thepresent invention, a protein of the invention may include variousstructural forms of the primary protein which retain biologicalactivity.

[0061] Broadly stated, the present invention provides an isolatedprotein containing a tyrosine phosphatase domain and which is associatedwith Lafora's disease.

[0062] In a preferred embodiment of the invention, the protein has theamino acid sequence as shown in SEQ ID NO:2 and FIG. 14. In anotherembodiment, the protein has the amino acid sequence shown inSEQ.ID.NO.:4 (or FIG. 8) or SEQ.ID.NO.: 6 (or FIG. 10).

[0063] In addition to full length amino acid sequences the proteins ofthe present invention also include truncations of the protein, andanalogs, and homologs of the protein and truncations thereof asdescribed herein. Truncated proteins may comprise peptides of at leastfifteen amino acid residues.

[0064] Analogs of the protein having the amino acid sequence shown inSEQ.ID.NO.:2 (FIG. 14) or SEQ.ID.NO.:4 (FIG. 8) or SEQ.ID.NO.:6 (FIG.10) and/or truncations thereof as described herein, may include, but arenot limited to an amino acid sequence containing one or more amino acidsubstitutions, insertions, and/or deletions. Amino acid substitutionsmay be of a conserved or non-conserved nature. Conserved amino acidsubstitutions involve replacing one or more amino acids of the proteinsof the invention with amino acids of similar charge, size, and/orhydrophobicity characteristics. When only conserved substitutions aremade the resulting analog should be functionally equivalent.Non-conserved substitutions involve replacing one or more amino acids ofthe amino acid sequence with one or more amino acids which possessdissimilar charge, size, and/or hydrophobicity characteristics.

[0065] One or more amino acid insertions may be introduced into theamino acid sequences shown in SEQ.ID.NO.:2 (FIG. 14) or SEQ.ID.NO.:4(FIG. 8) or SEQ.ID.NO.:6 (FIG. 10). Amino acid insertions may consist ofsingle amino acid residues or sequential amino acids ranging from 2 to15 amino acids in length. For example, amino acid insertions may be usedto destroy target sequences so that the protein is no longer active.This procedure may be used in vivo to inhibit the activity of a proteinof the invention.

[0066] Deletions may consist of the removal of one or more amino acids,or discrete portions from the amino acid sequence shown in SEQ.ID.NO.:2(FIG. 14) or SEQ.ID.NO.:4 (FIG. 8) or SEQ.ID.NO.:6 (FIG. 10). Thedeleted amino acids may or may not be contiguous. The lower limit lengthof the resulting analog with a deletion mutation is about 10 aminoacids, preferably 100 amino acids.

[0067] Analogs of a protein of the invention may be prepared byintroducing mutations in the nucleotide sequence encoding the protein.Mutations in nucleotide sequences constructed for expression of analogsof a protein of the invention must preserve the reading frame of thecoding sequences. Furthermore, the mutations will preferably not createcomplementary regions that could hybridize to produce secondary mRNAstructures, such as loops or hairpins, which could adversely affecttranslation of the receptor mRNA.

[0068] Mutations may be introduced at particular loci by synthesizingoligonucleotides containing a mutant sequence, flanked by restrictionsites enabling ligation to fragments of the native sequence. Followingligation, the resulting reconstructed sequence encodes an analog havingthe desired amino acid insertion, substitution, or deletion.

[0069] Alternatively, oligonucleotide-directed site specific mutagenesisprocedures may be employed to provide an altered gene having particularcodons altered according to the substitution, deletion, or insertionrequired. Deletion or truncation of a protein of the invention may alsobe constructed by utilizing convenient restriction endonuclease sitesadjacent to the desired deletion. Subsequent to restriction, overhangsmay be filled in, and the DNA religated. Exemplary methods of making thealterations set forth above are disclosed by Sambrook et al (MolecularCloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor LaboratoryPress, 1989).

[0070] The proteins of the invention also include homologs of the aminoacid sequence shown in SEQ.ID.NO.:2 (FIG. 14) or SEQ.ID.NO.:4 (FIG. 8)or SEQ.ID.NO.:6 (FIG. 10) and/or truncations thereof as describedherein. Such homologs are proteins whose amino acid sequences arecomprised of amino acid sequences that hybridize under stringenthybridization conditions (see discussion of stringent hybridizationconditions herein) with a probe used to obtain a protein of theinvention. Preferably, homologs of a protein of the invention will havea tyrosine phosphatase region which is characteristic of the protein.

[0071] A homologous protein includes a protein with an amino acidsequence having at least 70%, preferably 80-90% identity with the aminoacid sequence as shown in SEQ.ID.NO.:2 (FIG. 14) or SEQ.ID.NO.:4 (FIG.8) or SEQ.ID.NO.:6 (FIG. 10).

[0072] The invention also contemplates isoforms of the proteins of theinvention. An isoform contains the same number and kinds of amino acidsas a protein of the invention, but the isoform has a different molecularstructure. The isoforms contemplated by the present invention are thosehaving the same properties as a protein of the invention as describedherein.

[0073] The present invention also includes a protein of the inventionconjugated with a selected protein, or a selectable marker protein (seebelow) to produce fusion proteins. Additionally, immunogenic portions ofa protein of the invention are within the scope of the invention.

[0074] The proteins of the invention (including truncations, analogs,etc.) may be prepared using recombinant DNA methods. Accordingly, thenucleic acid molecules of the present invention having a sequence whichencodes a protein of the invention may be incorporated in a known mannerinto an appropriate expression vector which ensures good expression ofthe protein. Possible expression vectors include but are not limited tocosmids, plasmids, or modified viruses (e.g. replication defectiveretroviruses, adenoviruses and adeno-associated viruses), so long as thevector is compatible with the host cell used. The expression vectors are“suitable for transformation of a host cell”, means that the expressionvectors contain a nucleic acid molecule of the invention and regulatorysequences selected on the basis of the host cells to be used forexpression, which is operatively linked to the nucleic acid molecule.Operatively linked is intended to mean that the nucleic acid is linkedto regulatory sequences in a manner which allows expression of thenucleic acid.

[0075] The invention therefore contemplates a recombinant expressionvector of the invention containing a nucleic acid molecule of theinvention, or a fragment thereof, and the necessary regulatory sequencesfor the transcription and translation of the inserted protein-sequence.Suitable regulatory sequences may be derived from a variety of sources,including bacterial, fungal, or viral genes (For example, see theregulatory sequences described in Goeddel, Gene Expression Technology:Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990).Selection of appropriate regulatory sequences is dependent on the hostcell chosen, and may be readily accomplished by one of ordinary skill inthe art. Examples of such regulatory sequences include: atranscriptional promoter and enhancer or RNA polymerase bindingsequence, a ribosomal binding sequence, including a translationinitiation signal. Additionally, depending on the host cell chosen andthe vector employed, other sequences, such as an origin of replication,additional DNA restriction sites, enhancers, and sequences conferringinducibility of transcription may be incorporated into the expressionvector. It will also be appreciated that the necessary regulatorysequences may be supplied by the native protein and/or its flankingregions.

[0076] The invention further provides a recombinant expression vectorcomprising a DNA nucleic acid molecule of the invention cloned into theexpression vector in an antisense orientation. That is, the DNA moleculeis operatively linked to a regulatory sequence in a manner which allowsfor expression, by transcription of the DNA molecule, of an RNA moleculewhich is antisense to a nucleotide sequence comprising the nucleotidesas shown SEQ.ID.NO.:1, SEQ.ID.NO.:3 or SEQ.ID.NO.:5. Regulatorysequences operatively linked to the antisense nucleic acid can be chosenwhich direct the continuous expression of the antisense RNA molecule.

[0077] The recombinant expression vectors of the invention may alsocontain a selectable marker gene which facilitates the selection of hostcells transformed or transfected with a recombinant molecule of theinvention. Examples of selectable marker genes are genes encoding aprotein such as G418 and hygromycin which confer resistance to certaindrugs, 1-galactosidase, chloramphenicol acetyltransferase, or fireflyluciferase. Transcription of the selectable marker gene is monitored bychanges in the concentration of the selectable marker protein such asβ-galactosidase, chloramphenicol acetyltransferase, or fireflyluciferase. If the selectable marker gene encodes a protein conferringantibiotic resistance such as neomycin resistance transformant cells canbe selected with G418. Cells that have incorporated the selectablemarker gene will survive, while the other cells die. This makes itpossible to visualize and assay for expression of recombinant expressionvectors of the invention and in particular to determine the effect of amutation on expression and phenotype. It will be appreciated thatselectable markers can be introduced on a separate vector from thenucleic acid of interest.

[0078] The recombinant expression vectors may also contain genes whichencode a fusion moiety which provides increased expression of therecombinant protein; increased solubility of the recombinant protein;and aid in the purification of a target recombinant protein by acting asa ligand in affinity purification. For example, a proteolytic cleavagesite may be added to the target recombinant protein to allow separationof the recombinant protein from the fusion moiety subsequent topurification of the fusion protein.

[0079] Recombinant expression vectors can be introduced into host cellsto produce a transformant host cell. The term “transformant host cell”is intended to include prokaryotic and eukaryotic cells which have beentransformed or transfected with a recombinant expression vector of theinvention. The terms “transformed with”, “transfected with”,“transformation” and “transfection” are intended to encompassintroduction of nucleic acid (e.g. a vector) into a cell by one of manypossible techniques known in the art. Prokaryotic cells can betransformed with nucleic acid by, for example, electroporation orcalcium-chloride mediated transformation. Nucleic acid can be introducedinto mammalian cells via conventional techniques such as calciumphosphate or calcium chloride co-precipitation, DEAE-dextran-mediatedtransfection, lipofectin, electroporation or microinjection. Suitablemethods for transforming and transfecting host cells can be found inSambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition,Cold Spring Harbor Laboratory press (1989)), and other laboratorytextbooks.

[0080] Suitable host cells include a wide variety of prokaryotic andeukaryotic host cells. For example, the proteins of the invention may beexpressed in bacterial cells such as E. coli, insect cells (usingbaculovirus), yeast cells or mammalian cells. Other suitable host cellscan be found in Goeddel, Gene Expression Technology: Methods inEnzymology 185, Academic Press, San Diego, Calif. (1991).

[0081] The proteins of the invention may also be prepared by chemicalsynthesis using techniques well known in the chemistry of proteins suchas solid phase synthesis (Merrifield, 1964, J. Am. Chem. Assoc.85:2149-2154) or synthesis in homogenous solution (Houbenweyl, 1987,Methods of Organic Chemistry, ed. E. Wansch, Vol. 15 I and II, Thieme,Stuttgart).

[0082] III. Applications

[0083] A. Diagnostic Applications

[0084] As previously mentioned, the present inventors have isolated andsequenced a novel gene EPM2A and have shown that it is deleted ormutated in people with Lafora's disease. As a result, the presentinvention also includes a method of detecting Lafora's disease bydetecting a mutation or deletion in the Lafora's disease gene orprotein.

[0085] i) Detecting Mutations in the Nucleic Acid Sequence

[0086] In one embodiment, the present invention provides a method fordetecting Lafora's disease comprising detecting a deletion or mutationin the Lafora's disease gene in a sample obtained from an animal,preferably a mammal, more preferably a human. Preferably, the inventionprovides a method of detecting Lafora's disease comprising detecting adeletion or mutation in the Lafora's disease gene in the region betweenmarkers D6S1003 and D6S1042.

[0087] The Examples and Tables 1 to 3 summarize some of the mutationsfound in EPM2A in patient's with Lafora's Disease. Screening assays canbe developed for each of the mutations. Details of screening assays thatmay be employed for the 3 common mutations are provided in Example 3.

[0088] One of the common EPM2A mutations is a C→T nonsense mutation ofthe second base pair of exon 4 found at position 721 in FIG. 13. Thismutation destroys the recognition site for the restriction enzymeHaeIII. Accordingly, the C to T mutation can be detected in a sample bya method comprising:

[0089] (a) amplifying the nucleic acid sequences in the sample withprimers H1F (5′-GAATGCTCTTTCCACTTTGC-3) and PTPR(5′-GGCTCCTTAGGGAAATCAG-3′) in a polymerase chain reaction;

[0090] (b) digesting the amplified sequences with the restrictionendonuclease HaeIII; and

[0091] (c) determining the size of the digested sequences wherein thepresence of a fragment of approximately 199 bp indicates the sample isfrom an animal with Lafora's disease or an animal that is a carrier ofLafora's disease.

[0092] Another common mutation in EMP2A is a G→A mutation of base pair115 in exon 4 (position 836 in FIG. 13). This mutation creates a newPstI restriction site in the 520 bp DNA fragment that is amplified byprimers H1F and PTPR, which is not found in normal, non-carrierindividuals. Consequently, the present invention provides a method fordetecting a G to A mutation in EMP2A by a method comprising:

[0093] (a) amplifying the nucleic acid sequences in the sample withprimers H1F (5′-GAATGCTCTTTCCACTTTGC-3) and PTPR(5′-GGCTCCTTAGGGAAATCAG-3′) in a polymerase chain reaction;

[0094] (b) digesting the amplified sequences with the restrictionendonuclease PstI; and

[0095] (c) determining the size of the digested sequences wherein thepresence of at least one fragment of approximately 520 bp indicates thatthe sample is from an animal that does not have Lafora's disease or ananimal that is a carrier of Lafora's disease. Persons with Lafora'sdisease will have two variant bands of 195 base pairs and 350 basepairs.

[0096] Many families with Lafora's disease have deletions of EPM2A.Patients homozygous for these deletions can be detected by the absenceof PCR amplification products using primers JRGXBF/JRGXBR which amplifythe deleted region. Consequently, the present invention includes amethod for determining a deletion in the EMP2A gene by a methodcomprising:

[0097] (a) amplifying the nucleic acid sequences in the sample withprimers JRGXBF (5-TCCATTGTGCTAATGCTATCTC-3′) a nd JRGXBR(5′-TCAGCTTGCTTTGAGGATATTT-3′) in a polymerase chain reaction; and

[0098] (b) detecting amplified sequence wherein the absence of anamplified sequence indicates that the sample is from an animal withLafora's disease.

[0099] One skilled in the art will appreciate that other methods, inaddition to the ones discussed above and in the examples, can be used todetect mutations in the EPM2A gene. For example, in order to isolatenucleic acids from the Lafora's disease gene in a sample, one canprepare nucleotide probes from the nucleic acid sequences of theinvention. In addition, the nucleic acid probes described herein (forexample, see FIG. 1) can also be used. A nucleotide probe may belabelled with a detectable marker such as a radioactive label whichprovides for an adequate signal and has sufficient half life such as³²P, ³H, ¹⁴C or the like. Other detectable markers which may be usedinclude antigens that are recognized by a specific labelled antibody,fluorescent compounds, enzymes, antibodies specific for a labelledantigen, and chemiluminescent compounds. An appropriate label may beselected having regard to the rate of hybridization and binding of theprobe to the nucleotide to be detected and the amount of nucleotideavailable for hybridization.

[0100] Accordingly, the present invention also relates to a method ofdetecting the presence of a nucleic acid molecule from the EPM2A gene ina sample comprising contacting the sample under hybridization conditionswith one or more of nucleotide probes which hybridize to the nucleicacid molecules and are labelled with a detectable marker, anddetermining the degree of hybridization between the nucleic acidmolecule in the sample and the nucleotide probes. Preferably, thenucleic acid probes hybridize with a portion of the EPM2A genecontaining a mutation site in Lafora's disease, for example, in theregion between marker DS61003 and DS61042.

[0101] Hybridization conditions which may be used in the methods of theinvention are known in the art and are described for example in SambrookJ, Fritch E F, Maniatis T. In: Molecular Cloning, A Laboratory Manual,1989. (Nolan C, Ed.), Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. The hybridization product may be assayed using techniquesknown in the art. The nucleotide probe may be labelled with a detectablemarker as described herein and the hybridization product may be assayedby detecting the detectable marker or the detectable change produced bythe detectable marker.

[0102] Prior to hybridizing a sample with DNA probes, the sample can betreated with primers that flank the EPM2A gene in order to amplify thenucleic acid sequences in the sample. The primers used may be the onesdescribed in the present application. For example, primers specific forthe transcript A include 266F and GSP3. Primers for the transcript Binclude AA490925F and AA490925R. In addition, the sequence of the EPM2Agene provided herein also permits the identification and isolation, orsynthesis of new nucleotide sequences which may be used as primers toamplify a nucleic acid molecule of the invention, for example in thepolymerase chain reaction (PCR) which is discussed in more detail below.The primers may be used to amplify the genomic DNA of other species. ThePCR amplified sequences can be examined to determine the relationshipbetween the genes of various species.

[0103] The length and bases of the primers for use in the PCR areselected so that they will hybridize to different strands of the desiredsequence and at relative positions along the sequence such that anextension product synthesized from one primer when it is separated fromits template can serve as a template for extension of the other primerinto a nucleic acid of defined length. Primers which may be used in theinvention are oligonucleotides i.e. molecules containing two or moredeoxyribonucleotides of the nucleic acid molecule of the invention whichoccur naturally as in a purified restriction endonuclease digest or areproduced synthetically using techniques known in the art such as forexample phosphotriester and phosphodiester methods (See Good et al Nucl.Acid Res 4:2157, 1977) or automated techniques (See for example,Conolly, B. A. Nucleic Acids Res. 15:15(7): 3131, 1987). The primers arecapable of acting as a point of initiation of synthesis when placedunder conditions which permit the synthesis of a primer extensionproduct which is complementary to the DNA sequence of the invention i.e.in the presence of nucleotide substrates, an agent for polymerizationsuch as DNA polymerase and at suitable temperature and pH. Preferably,the primers are sequences that do not form secondary structures by basepairing with other copies of the primer or sequences that form a hairpin configuration. The primer preferably contains between about 7 and 25nucleotides.

[0104] The primers may be labelled with detectable markers which allowfor detection of the amplified products. Suitable detectable markers areradioactive markers such as P-32, S-35, I-125, and H-3, luminescentmarkers such as chemiluminescent markers, preferably luminol, andfluorescent markers, preferably dansyl chloride,fluorcein-5-isothiocyanate, and 4-fluor-7-nitrobenz-2-axa-1,3 diazole,enzyme markers such as horseradish peroxidase, alkaline phosphatase,β-galactosidase, acetylcholinesterase, or biotin.

[0105] It will be appreciated that the primers may containnon-complementary sequences provided that a sufficient amount of theprimer contains a sequence which is complementary to a nucleic acidmolecule of the invention or oligonucleotide fragment thereof, which isto be amplified. Restriction site linkers may also be incorporated intothe primers allowing for digestion of the amplified products with theappropriate restriction enzymes facilitating cloning and sequencing ofthe amplified product.

[0106] In an embodiment of the invention a method of determining thepresence of a nucleic acid molecule of the invention is providedcomprising treating the sample with primers which are capable ofamplifying the nucleic acid molecule or a predetermined oligonucleotidefragment thereof in a polymerase chain reaction to form amplifiedsequences, under conditions which permit the formation of amplifiedsequences and, assaying for amplified sequences.

[0107] The polymerase chain reaction refers to a process for amplifyinga target nucleic acid sequence as generally described in Innis et al,Academic Press, 1990 in Mullis et al., U.S. Pat. No. 4,863,195 andMullis, U.S. Pat. No. 4,683,202 which are incorporated herein byreference. Conditions for amplifying a nucleic acid template aredescribed in M.A. Innis and D.H. Gelfand, PCR Protocols, A Guide toMethods and Applications M.A. Innis, D.H. Gelfand, J.J. Sninsky and T.J.White eds, pp3-12, Academic Press 1989, which is also incorporatedherein by reference.

[0108] The amplified products can be isolated and distinguished based ontheir respective sizes using techniques known in the art. For example,after amplification, the DNA sample can be separated on an agarose geland visualized, after staining with ethidium bromide, under ultra violet(UW) light. DNA may be amplified to a desired level and a furtherextension reaction may be performed to incorporate nucleotidederivatives having detectable markers such as radioactive labelled orbiotin labelled nucleoside triphosphates. The primers may also belabelled with detectable markers as discussed above. The detectablemarkers may be analyzed by restriction and electrophoretic separation orother techniques known in the art.

[0109] The conditions which may be employed in the methods of theinvention using PCR are those which permit hybridization andamplification reactions to proceed in the presence of DNA in a sampleand appropriate complementary hybridization primers. Conditions suitablefor the polymerase chain reaction are generally known in the art. Forexample, see M. A. Innis and D. H. Gelfand, PCR Protocols, A guide toMethods and Applications M. A. Innis, D. H. Gelfand, J. J. Sninsky andT. J. White eds, pp3-12, Academic Press 1989, which is incorporatedherein by reference. Preferably, the PCR utilizes polymerase obtainedfrom the thermophilic bacterium Thermus aquatics (Taq polymerase,GeneAmp Kit, Perkin Elmer Cetus) or other thermostable polymerase may beused to amplify DNA template strands.

[0110] It will be appreciated that other techniques such as the LigaseChain Reaction (LCR) and NASBA may be used to amplify a nucleic acidmolecule of the invention (Barney in “PCR Methods and Applications”,August 1991, Vol.1(1), page 5, and European Published Application No.0320308, published Jun. 14, 1989, and U.S. Serial NO. 5,130,238 toMalek).

[0111] (ii) Detecting the Laforin Protein

[0112] In another embodiment, the present invention provides a methodfor detecting Lafora's disease comprising determining if the Laforinprotein is present in a sample from an animal.

[0113] The Laforin protein of the present invention may be detected in abiological sample using antibodies that are specific for Laforin usingvarious immunoassays that are discussed below.

[0114] Conventional methods can be used to prepare the antibodies. Forexample, by using a peptide from the Laforin protein of the invention,polyclonal antisera or monoclonal antibodies can be made using standardmethods. A mammal, (e.g., a mouse, hamster, or rabbit) can be immunizedwith an immunogenic form of the peptide which elicits an antibodyresponse in the mammal. Techniques for conferring immunogenicity on apeptide include conjugation to carriers or other techniques well knownin the art. For example, the peptide can be administered in the presenceof adjuvant. The progress of immunization can be monitored by detectionof antibody titers in plasma or serum. Standard ELISA or otherimmunoassay procedures can be used with the immunogen as antigen toassess the levels of antibodies. Following immunization, antisera can beobtained and, if desired, polyclonal antibodies isolated from the sera.

[0115] To produce monoclonal antibodies, antibody producing cells(lymphocytes) can be harvested from an immunized animal and fused withmyeloma cells by standard somatic cell fusion procedures thusimmortalizing these cells and yielding hybridoma cells. Such techniquesare well known in the art, (e.g., the hybridoma technique originallydeveloped by Kohler and Milstein (Nature 256, 495497 (1975)) as well asother techniques such as the human B-cell hybridoma technique (Kozbor etal., Immunol. Today 4, 72 (1983)), the EBV-hybridoma technique toproduce human monoclonal antibodies (Cole et al. Monoclonal Antibodiesin Cancer Therapy (1985) Allen R Bliss, Inc., pages 77-96), andscreening of combinatorial antibody libraries (Huse et al., Science 246,1275 (1989)]. Hybridoma cells can be screened immunochemically forproduction of antibodies specifically reactive with the peptide and themonoclonal antibodies can be isolated. Therefore, the invention alsocontemplates hybridoma cells secreting monoclonal antibodies withspecificity for a protein of the invention.

[0116] The term “antibody” as used herein is intended to includefragments thereof which also specifically react with a protein, of theinvention, or peptide thereof. Antibodies can be fragmented usingconventional techniques and the fragments screened for utility in thesame manner as described above. For example, F(ab′)₂ fragments can begenerated by treating antibody with pepsin. The resulting F(ab′)₂fragment can be treated to reduce disulfide bridges to produce Fab′fragments.

[0117] Chimeric antibody derivatives, i.e., antibody molecules thatcombine a non-human animal variable region and a human constant regionare also contemplated within the scope of the invention. Chimericantibody molecules can include, for example, the antigen binding domainfrom an antibody of a mouse, rat, or other species, with human constantregions. Conventional methods may be used to make chimeric antibodiescontaining the immunoglobulin variable region which recognizes a CipAprotein (See, for example, Morrison et al., Proc. Natl. Acad. Sci.U.S.A. 81,6851 (1985); Takeda et al., Nature 314, 452 (1985), Cabilly etal., U.S. Pat. No. 4,816,567; Boss et al., U.S. Pat. No. 4,816,397;Tanaguchi et al., European Patent Publication EP171496; European PatentPublication 0173494, United Kingdom patent GB 2177096B).

[0118] Monoclonal or chimeric antibodies specifically reactive with aprotein of the invention as described herein can be further humanized byproducing human constant region chimeras, in which parts of the variableregions, particularly the conserved framework regions of theantigen-binding domain, are of human origin and only the hypervariableregions are of non-human origin. Such immunoglobulin molecules may bemade by techniques known in the art, (e.g., Teng et al., Proc. Natl.Acad. Sci. U.S.A., 80, 7308-7312 (1983); Kozbor et al., ImmunologyToday, 4, 7279 (1983); Olsson et al., Meth. Enzymol., 92, 3-16 (1982)),and PCT Publication WO92/06193 or EP 0239400). Humanized antibodies canalso be commercially produced (Scotgen Limited, 2 Holly Road,Twickenham, Middlesex, Great Britain.)

[0119] Specific antibodies, or antibody fragments, reactive against aprotein of the invention may also be generated by screening expressionlibraries encoding immunoglobulin genes, or portions thereof, expressedin bacteria with peptides produced from the nucleic acid molecules ofthe present invention. For example, complete Fab fragments, VH regionsand FV regions can be expressed in bacteria using phage expressionlibraries (See for example Ward et al., Nature 341, 544-546: (1989);Huse et al., Science 246, 1275-1281 (1989); and McCafferty et al. Nature348, 552-554 (1990)).

[0120] Antibodies may also be prepared using DNA immunization. Forexample, an expression vector containing a nucleic acid of the invention(as described above) may be injected into a suitable animal such asmouse. The protein of the invention will therefore be expressed in vivoand antibodies will be induced. The antibodies can be isolated andprepared as described above for protein immunization.

[0121] The antibodies may be labelled with a detectable marker includingvarious enzymes, fluorescent materials, luminescent materials andradioactive materials. Examples of suitable enzymes include horseradishperoxidase, biotin, alkaline phosphatase, β-galactosidase, oracetylcholinesterase; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; and examples ofsuitable radioactive material include S-35, Cu-64, Ga-67, Zr-89, Ru-97,Tc-99m, Rh-105, Pd-109, In-111, I-123, I-125, I131, Re-186, Au-198,Au-199, Pb-203, At-211, Pb-212 and Bi-212. The antibodies may also belabelled or conjugated to one partner of a ligand binding pair.Representative examples include avidin-biotin and riboflavin-riboflavinbinding protein. Methods for conjugating or labelling the antibodiesdiscussed above with the representative labels set forth above may bereadily accomplished using conventional techniques.

[0122] The antibodies reactive against proteins of the invention (e.g.enzyme conjugates or labelled derivatives) may be used to detect aprotein of the invention in various samples, for example they may beused in any known immunoassays which rely on the binding interactionbetween an antigenic determinant of a protein of the invention and theantibodies. Examples of such assays are radioimmunoassays, enzymeimmunoassays (e.g. ELISA), immunofluorescence, immuno-precipitation,latex agglutination, hemagglutination, and histochemical tests. Thus,the antibodies may be used to identify or quantify the amount of aprotein of the invention in a sample in order to diagnose the presenceof Lafora's disease.

[0123] In a method of the invention a predetermined amount of a sampleor concentrated sample is mixed with antibody or labelled antibody. Theamount of antibody used in the process is dependent upon the labellingagent chosen. The resulting protein bound to antibody or labelledantibody may be isolated by conventional isolation techniques, forexample, salting out, chromatography, electrophoresis, gel filtration,fractionation, absorption, polyacrylamide gel electrophoresis,agglutination, or combinations thereof.

[0124] The sample or antibody may be insolubilized, for example, thesample or antibody can be reacted using known methods with a suitablecarrier. Examples of suitable carriers are Sepharose or agarose beads.When an insolubilized sample or antibody is used protein bound toantibody or unreacted antibody is isolated by washing. For example, whenthe sample is blotted onto a nitrocellulose membrane, the antibody boundto a protein of the invention is separated from the unreacted antibodyby washing with a buffer, for example, phosphate buffered saline (PBS)with bovine serum albumin (BSA).

[0125] When labelled antibody is used, the presence of Laforin can bedetermined by measuring the amount of labelled antibody bound to aprotein of the invention in the sample or of the unreacted labelledantibody. The appropriate method of measuring the labelled material isdependent upon the labelling agent.

[0126] When unlabelled antibody is used in the method of the invention,the presence of Laforin can be determined by measuring the amount ofantibody bound to the protein using substances that interactspecifically with the antibody to cause agglutination or precipitation.In particular, labelled antibody against an antibody specific for aprotein of the invention, can be added to the reaction mixture. Thepresence of a protein of the invention can be determined by a suitablemethod from among the already described techniques depending on the typeof labelling agent. The antibody against an antibody specific for aprotein of the invention can be prepared and labelled by conventionalprocedures known in the art which have been described herein. Theantibody against an antibody specific for a protein of the invention maybe a species specific anti-immunoglobulin antibody or monoclonalantibody, for example, goat anti-rabbit antibody may be used to detectrabbit antibody specific for a protein of the invention.

[0127] (iii) Kits

[0128] The reagents suitable for carrying out the methods of theinvention may be packaged into convenient kits providing the necessarymaterials, packaged into suitable containers. Such kits may include allthe reagents required to detect a nucleic acid molecule or protein ofthe invention in a sample by means of the methods described herein, andoptionally suitable supports useful in performing the methods of theinvention.

[0129] In one embodiment of the invention, the kit includes primerswhich are capable of amplifying a nucleic acid molecule of the inventionor a predetermined oligonucleotide fragment thereof, all the reagentsrequired to produce the amplified nucleic acid molecule or predeterminedfragment thereof in the polymerase chain reaction, and means forassaying the amplified sequences. The kit may also include restrictionenzymes to digest the PCR products. In another embodiment of theinvention the kit contains a nucleotide probe which hybridizes with anucleic acid molecule of the invention, reagents required forhybridization of the nucleotide probe with the nucleic acid molecule,and directions for its use. In a further embodiment of the invention thekit includes antibodies of the invention and reagents required forbinding of the antibody to a protein of the invention in a sample.

[0130] The methods and kits of the present invention may be used todetect Lafora's disease. Samples which may be tested include bodilymaterials such as blood, urine, serum, tears, saliva, feces, tissues,cells and the like. In addition to human samples, samples may be takenfrom mammals such as non-human primates, etc.

[0131] Before testing a sample in accordance with the methods describedherein, the sample may be concentrated using techniques known in theart, such as centrifugation and filtration. For the hybridization and/orPCR-based methods described herein, nucleic acids may be extracted fromcell extracts of the test sample using techniques known in the art.

[0132] B. Therapeutic Applications

[0133] As mentioned previously, the nucleic acid molecules of thepresent invention are deleted or mutated in people with Lafora'sdisease. Accordingly, the present invention provides a method oftreating or preventing Lafora's disease by administering a nucleic acidsequence containing a sufficient portion of the EPM2A gene to treat orprevent Lafora's disease.

[0134] Recombinant molecules comprising a nucleic acid sequence orfragment thereof, may be directly introduced into cells or tissues invivo using delivery vehicles such as retroviral vectors, adenoviralvectors and DNA virus vectors. They may also be introduced into cells invivo using physical techniques such as microinjection andelectroporation or chemical methods such as coprecipitation andincorporation of DNA into liposomes. Recombinant molecules may also bedelivered in the form of an aerosol or by lavage.

[0135] The nucleic acid sequences may be formulated into pharmaceuticalcompositions for adminstration to subjects in a biologically compatibleform suitable for administration in vivo. By “biologically compatibleform suitable for administration in vivo” is meant a form of thesubstance to be administered in which any toxic effects are outweighedby the therapeutic effects. The substances may be administered to livingorganisms including humans, and animals. Administration of atherapeutically active amount of the pharmaceutical compositions of thepresent invention is defined as an amount effective, at dosages and forperiods of time necessary to achieve the desired result. For example, atherapeutically active amount of a substance may vary according tofactors such as the disease state, age, sex, and weight of theindividual, and the ability of antibody to elicit a desired response inthe individual. Dosage regima may be adjusted to provide the optimumtherapeutic response. For example, several divided doses may beadministered daily or the dose may be proportionally reduced asindicated by the exigencies of the therapeutic situation.

[0136] The active substance may be administered in a convenient mannersuch as by injection (subcutaneous, intravenous, etc.), oraladministration, inhalation, transdermal application, or rectaladministration. Depending on the route of administration, the activesubstance may be coated in a material to protect the compound from theaction of enzymes, acids and other natural conditions which mayinactivate the compound.

[0137] The compositions described herein can be prepared by per se knownmethods for the preparation of pharmaceutically acceptable compositionswhich can be administered to subjects, such that an effective quantityof the active substance is combined in a mixture with a pharmaceuticallyacceptable vehicle. Suitable vehicles are described, for example, inRemington's Pharmaceutical Sciences (Remington's PharmaceuticalSciences, Mack Publishing Company, Easton, Pa., USA 1985). On thisbasis, the compositions include, albeit not exclusively, solutions ofthe substances in association with one or more pharmaceuticallyacceptable vehicles or diluents, and contained in buffered solutionswith a suitable pH and iso-osmotic with the physiological fluids.

[0138] C. Experimental Models

[0139] The present invention also includes methods and experimentalmodels for studying the function of the EPM2A gene and Laforin protein.Cells, tissues and non-human animals that lack the EPM2A gene orpartially lack in Laforin expression may be developed using recombinantexpression vectors having a specific deletion or mutation in the EPM2Agene. A recombinant expression vector may be used to inactivate or alterthe EPM2A gene by homologous recombination and thereby create an EPM2Adeficient cell, tissue or animal.

[0140] Null alleles may be generated in cells, such as embryonic stemcells by deletion mutation. A recombinant EPM2A gene may also beengineered to contain an insertion mutation which inactivates EPM2A.Such a construct may then be introduced into a cell, such as anembryonic stem cell, by a technique such as transfection,electroporation, injection etc. Cells lacking an intact EPM2A gene maythen be identified, for example by Southern blotting, Northern Blottingor by assaying for EPM2A using the methods described herein. Such cellsmay then be fused to embryonic stem cells to generate transgenicnon-human animals deficient in EPM2A. Germline transmission of themutation may be achieved, for example, by aggregating the embryonic stemcells with early stage embryos, such as 8 cell embryos, in vitro;transferring the resulting blastocysts into recipient females and;generating germline transmission of the resulting aggregation chimeras.Such a mutant animal may be used to define specific cell populations,developmental patterns and in vivo processes, normally dependent onEPM2A expression. The present invention also includes the preparation oftissue specific knock-outs of the EPM2A gene.

[0141] The following non-limiting examples are illustrative of thepresent invention:

EXAMPLES Example 1

[0142] Characterization of EPM2A

[0143] Materials and Methods

[0144] Patients. The diagnosis of Lafora's disease in patients withteenage onset progressive myoclonus epilepsy was confirmed bydemonstration of Lafora bodies in skin, liver, muscle or brain biopsies(6-9) in at least one affected member from each of 38 families includedin this study.

[0145] Physical mapping. Using mapping data available from the WhiteheadInstitute/MIT Genome Center (http://mit-genome.wi.mit.edu/) as well asby identifying additional clones it was possible to establish anoverlapping set of yeast artificial chromosome (YAC) clones betweenD6S1003 and D6S311. A total of 136 markers (12 genes, 41 ESTs, and 83STSs/probes) were assayed against the YAC contig and 32 of these werefound to be in the EPM2A critical region (FIG. 1). We also isolated 129P1-derived artificial chromosomes (PACs) which cover an estimated 90% ofthe region between D6S1003 and D6S311 and have aligned the PACs by probecontent, restriction mapping, as well fingerprint analysis. Informationon all DNA markers can be found at the Genome DataBase(http://www.gdbwww.gdb.org/) or the Sanger Genome Center WWW site(http://www.sanger.ac.uk/HGP/Chr6/).

[0146]FIG. 1 illustrates the physical map of the Lafora's diseasecritical region. (A). A yeast artificial chromosome (YAC) contig wasestablished covering the 1.5 Mb critical region between D6S1003 andD6S311. The presence of a DNA marker on a YAC clone is shown by acorresponding vertical bar. The markers that are highlighted with acircle and a square represent genetic markers or ESTs, respectively,while the remaining ones are unique landmarks (STSs). The region betweenD6S1003 and D6S1042 that demonstrated an extended region of homozygosityin affected members of a previously uncharacterized family is shown by athicker horizontal bar and this is the new EPM2A critical region (seeFIG. 2A); (B). A P1-derived artificial chromosome (PAC) map encompassingthe immediate region surround D6S1703. The extent of the deletion couldbe defined by PCR analysis of mapped STSs (see FIG. 2B). LDCR4represents a transcript of unidentified function and EPM2A is the Laforadisease gene. Since the 5′-end of this gene is not yet known it isrepresented with a dashed line.

[0147] Northern blots, cDNA library screening, and RACE. Multiple-tissue(cat. #7760-1) and Human Brain II (cat. #7755-1) Northern blots werepurchased from Clontech and hybridization was carried out as recommendedby the supplier. The transcript A specific probe was generated using PCRprimers 266F (5′-CGGCACGAGGATTATTCAAG-3′) and GSP3(5′-GCTCGGGTACTGAGGTCTG-3′) which amplified an 190 bp fragment from cDNAclone 266552 (FIG. 3). The transcript B specific probe was derived usingPCR primers AA490925F (5′-AGTTGTTACACAGGGTTGTTGG-3′) and AA490925R(5′-AGGCTGTACATCAGACAGAAGG-3′) which amplified an 373 bp segment fromcDNA SFB14 (FIG. 3). We have sequenced the HTF-island shown in FIG. 1Bat the 5′-end of EPM2A.

[0148] Genotyping. Haplotypes for 6q23-25 were constructed for allfamily members using microsatellite markers at loci D6S314, D6S1704,D6S1003, D6S1010, D6S1049, D6S1703, D6S1042, D6S1649, D6S978, D6S311 andD6S1637. Primer sequences were obtained from Genethon or from theCooperative Human Linkage Centre. PCR conditions have been reportedpreviously (13). PCR products were separated on polyacrylamide gels. In8 families (20%), haplotype analyses revealed evidence against linkageto 6q23-25. Of the remaining 30 LD families 16 reported a history ofconsanguinity. Thirty-one of these families have been describedpreviously (refs. 12, 13, 25, 25).

[0149] Mutation Analysis. Mutations were detected by radioactive cyclesequencing using the Thermosequenase Kit (Amersham Life Science) withQiagen column purified PCR products. The combinations of PCR primerpairs used were JRGXBCF (5′-TCCATTGTGCTAATGCTATCTC-3′) and JRGXBCR(5′-TCAGCTTGCTTTGAGGATAM-3′); product size 310 bp, 824F(5′-GCCGAGTACAGATGCTGCC-3′ and 824R (5′-CACACAGTCCTTTCAGTTCAGG-3′);product size 384 bp, and H1F (5′-GAATGCTCTTTCCACTTT GC-3′ and 824R;product size 587 bp. The position of the primers are shown in FIG. 3.

[0150] Characterization of Lafora's Disease Gene

[0151] To characterize the extent of the homozygous deletion in theaffected in LD-L4 a P1-derived artificial chromosome (PAC) contigextending outwards from D6S1703 was constructed. It could be determinedthat the deletion encompassed approximately 50 kb and that it did notinterrupt directly the LDCR4 transcription unit (FIG. 1B). PAC clones365C1, 466P17 and 28H5 (which encompassed the deletion) were sequencedin order to identify new candidate transcription units (FIG. 1B). Asegment of DNA (E42) located within the deletion detected a single EST(clone 743381) in the database (FIG. 3). DNA sequencing of this cDNAindicated it contained a segment of identity with one other EST(266552). This EST, however, was aligned previously with others intoseparate groups (or Unigenes named Hs.22464 and Hs.112229).Subsequently, we used clone 743381 and 824559 and PCR primers derivedfrom their sequence for screening of multiple cDNA libraries in anattempt to clone the entire coding region of this gene.

[0152]FIG. 2 shows a refined mapping of the Lafora disease gene. (A)Pedigrees and genotype data are provided for Lafora family LD39.Individuals affected (solid) or unaffected (open) with Lafora diseaseare indicated. Below each individual is the corresponding genotype data(the markers are listed in their order from centromere (top) to telomere(bottom) as determined using the physical map shown in FIG. 1). Theboxed segments of the haplotypes indicate regions of homozygosity. Theloci in bold indicate the previous LD critical region. (B) Detection of2 markers (D6S1703 and 109F4.E05.5) determined to be absent by PCR inthe affected members of the consanguineous Lafora family LD-L4.

[0153]FIG. 3 shows overlapping cDNA clones aligned with genomic DNAsegments. The portions of each cDNA clone for which there was sequenceis represented with a box. The corresponding genomic fragments are shownas stippled boxes below. The clones preceded with an (E) and (H)represent EcOR1 and HindIII fragments, respectively. The positions ofthe primers used for mutation screening are shown as is the site of thephosphatase domain and the stop codon (*).

[0154] Through analysis of the alignment of the DNA sequences of all ofthe EST clones as well as the newly identified cDNAs, at least 4putative types of transcripts that corresponded to EPM2A could bedefined (named transcript A, B, C, and D (FIG. 3). The cDNAs groupedinto transcript A could be categorized based on regions of sequenceidentity at their 3′-ends. A consensus sequence was compiled and it wasfound to be distributed amongst 4 exons spanning approximately 130 kb(FIGS. 1A and 3). A single cDNA (266552) representing transcript Bshared exact identity with transcript A except for the omission of a1,700 bp segment due to splicing (FIGS. 3 and 4). By comparing thecorresponding genomic regions to the cDNAs a common origin fortranscript A and B could be verified suggesting they are alternativeforms of the same gene, the gene-products, of which, would be predictedto have unique carboxyl-terminal amino acid sequences (FIG. 4B).

[0155]FIG. 4 shows the nucleotide sequence of cDNA encoding the EPM2Agene together with the predicted amino acid sequence. (A) The consensusnucleotide sequence was derived from the cDNA clones 266552, RACE-A,RACE-B, RACE-C, and RACE-D shown in FIG. 3. The position of themutations identified are indicated. The (*) indicates a stop mutationsite and the position of 2 known splice junctions is shown by thehorizontal arrows. An A to T polymorphism which is present inapproximately 40-50% of the population is shown; (B) the deduced Cterminus of transcript A compared with transcript B. The latter arisesdue to the removal by splicing of nt 738-2508 (FIG. 3 and FIG. 4A),which would be predicted to generate an isoform with a unique 3, end. Atthe present time, transcript B is known to extend to position 94 of thepredicted amino acid sequence shown (FIG. 4A). Transcript C (cDNA SFB14)is described elsewhere (C), the putative PTP active sites of EPM2A,MTM1, PTEN, PTP18, dPTP61F and viral PTP. The shaded amino acids (C andR) represent catalytic residues. On the basis of sequence analysisalone, laforin predicts an intracellular PTP with dual specificityphosphatase activity.

[0156] The inventors determined a partial map (FIG. 3) and sequenced thecorresponding genomic regions that contained nucleotide identity tothese segments to prove their common origin. The results suggest thattranscript A, B, C and D are indeed alternatively spliced forms of thesame gene. The consensus sequence presently compiled for transcript Awas distributed amongst at least 4 exons spanning greater than 50 kbwhile transcript B was represented as a contiguous segment of DNA. Asingle EST clone, 743381, which represents another alternatively splicedform that appeared to be most common to transcript A was also identified(FIG. 3A). It contained at least 8 exons (FIG. 3) but a significant openreading frame was not detected. The newly identified gene, EPM2A, whichencodes Laforin, was the only one determined to be deleted in familyLD-L4 (FIG. 1).

[0157] Two other single cDNA clones, SFB14 and 743381, which couldrepresent additional alternative forms of EPM2A, were also identified(FIG. 3). SFB14 was contiguous to genomic DNA and identical to the3‘-end of transcript A except it’s open reading frame (ORF) waspredicted to extend 48 amino acids 5′ into the last intron shown in FIG.3. Clone 743381 contained 8 exons with appropriate exon-intronboundaries (FIG. 3) but its significance could not be assessed due tothe lack of continuous open reading frame.

[0158] In addition to the essential cysteine and arginine residues foundin all PTPs (FIG. 4C), EPM2A contains an aspartic acid positioned 31residues amino-terminal of the cysteine nucleophile. This amino acid isimportant for catalysis as it is located on a loop that undergoesconformational change when substrate is bound to enzyme.

[0159] The corresponding mRNA for EPM2A was determined to be 3200nucleotides in length in multiple tissues based on RNA gel-blothybridization experiments. FIG. 5 shows RNA expression pattern of theLaforin gene. Northern blot analysis in different tissues as indicatedat the top. The probes used are described in the Materials and Methodsand the exposure time was 4 days at −80° C. The EPM2A message isobserved in all tissues tested and the apparent overexpression in heartand skeletal muscle is due to overloading of mRNA in these lanes as wasseen when using any gene-specific probe. The results of FIG. 5illustrate that strong hybridization signals were detected in skeletalmuscle RNA and clear signals were also seen in heart, brain, placenta,lung, liver, kidney and pancreas. In addition, the same size mRNA wasdetected in cerebellum, cerebral cortex, medulla, spinal cord, occipitalpole, frontal lobe, temporal lobe, and putamen. Identical resultsshowing the same 3200 nucleotide message and tissue distribution wereobserved when a DNA probe believed to be specific for each isoform ofthe gene based on the established consensus sequences, was used. Forexample, a probe derived from the 3′-UTR region of transcript B of EPM2Awas determined unequivocally to be specific for this isoform. Fortranscript A, the probe was generated from the unique region shown inFIG. 4A and RT-PCR experiments seemed to confirm the specificity of thisfragment (data not shown). On the basis of northern-blot results and therelative number of ESTs identified, it is probable that transcript Arepresents the major isoform of EPM2A, and that it corresponds to the3.2 kb mRNA. From the analysis of the genomic DNA sequence, we haveidentified an additional ORF at the HTF-island (FIG. 3). As thispredicted exon has all the proposed features of the consensus sequenceof a eukaryotic translation initiation site, and 113 nt of it arerepresented in the consensus cDNA sequence, it could represent the 5′end of EPM2A.

[0160] The protein encoded by EPM2A contains an amino acid motif (FIG.1C) that corresponds with the consensus sequence, HcxxGxxRS(T), of thecatalytic site of PTPs. In addition to the essential cysteine andarginine residues found in all PTPs (FIG. 4C), EPM2A contains theexpected aspartic acid necessary for completion of the catalyticreaction, positioned 31-aa N terminal of the cysteine nucleophile.

[0161] In an attempt to isolate the remainder of the coding region forthese transcripts we performed multiple rounds of 5′-RACE on total brainand poly(A)+ mRNA which has allowed us to extend transcript A (but nottranscript B) further. Beyond the most 5′-sequences shown in FIG. 4,however, all of the RACE clones recovered seemed to share the expectedDNA sequences but then diverged in different ways that did not allow fora common consensus to be established. However, comparative DNA sequenceanalysis of the human EPM2A gene its corresponding mouse homolog (alsocalled EPMA) confirmed the full length gene sequence as shown in FIG.13.

[0162] The deduced amino acid sequence of the newly identifiedprotein(s) indicated that transcripts A, B, C and D encode a 9 aminoacid motif (FIG. 4A) that corresponds exactly to the consensus sequence,HCxxGxxRS(T), of the active catalytic site of protein tyrosinephosphatases (PTPs) (14,15). So far, no other structural motifs could beidentified, and from the sequence it is not apparent if this proteinbelongs to the receptor-like PTPs, the intracellular PTPs, or the dualspecificity phosphatases (DSPs) which dephosphorylate both tyrosine andserine/threonine residues (16). The identification of the EPM2A gene asa putative PTP provides the first clue to understand the basic defect.

[0163] At the HTF-island shown in FIG. 3, we have identified throughGRAIL analysis (http://compbio.oml.gov) an additional putative exon 189nucleotides in length. An ATG (AUG) triplet is present at the beginningof this predicted ORF and the nucleotide sequence surrounding theconsensus sequence (CCCGCCAUGC) has the proposed features of theconsensus sequence (GCCA/GCCAUGG) of a eukaryotic translation initiationsite (12). The predicted start exon maintains open reading frame withthe most 5′ sequence of transcript A and this combined stretch of 298nucleotides contains exon/intron junction sequences with splice sitesthat confirm with the consensus in other mammalian genes. If thepredicted exon is part of EPM2A, transcript A would be predicted to be317 amino acids long.

Example 2

[0164] EPM2A Mutations

[0165] Using the available genomic structure for the gene, theinventors' screened an affected member from each of 30 Lafora familiesfor mutations by direct DNA sequencing. A total of 14 mutations weredetected consisting of 12 different DNA sequence alterations and 2microdeletions. The mutations are summarized in Table 3. The mutationfrom C to A at position-12 refers to a mutation that occurs 12 basesupstream from the ATG start codon in FIG. 13. Some of the sequenceupstream of the ATG is as follows:

[0166] . . . gcccgggtattcgcgccgCcgccgcccgccATG . . .

[0167] The mutation site at −12 is indicated with a capital C. To date,mutations have been found in 65% of EPM2A families. Some of themutations are discussed below.

[0168] Two mutations that, based on the current consensus sequences werespecific for transcript A, could be detected. Family LD-5 contained ahomozygous C to T point mutation which resulted in an arginine tocysteine change affecting a region of unknown function. To test for thepresence of the C to T point mutation in family LD-5 in the unaffectedpopulation PCR was completed on 54 samples (108 chromosomes) usingJRGXBF and JRGXBR primers and the product was blotted in duplicate. Onemembrane was hybridized with a wild type oligonucleotide(ATCATGACCGTTGCTGTAC) and the other with LD5 mutant(TCATCATGACTGTTGCTGTAC) oligonucleotide at 42° C. (washing with 5×SSC atroom temperature for 20 minutes followed by 2×SSC 20 minutes at 65° C.).No mutant alleles were found.

[0169] The inventors have screened 100 normal chromosomes for thischange and no mutant alleles were found. In family I-22 a homozygous Gto T non-sense change in a region specific to transcript A would predictpremature termination of the EPM2A protein. In sequences common to bothisoforms the inventors detected in the consanguineous family EPM2A00-4,a homozygous insertion of an A which would result in a frameshift thatwould cause an interruption of the tyrosine phosphatase domain. Theinventors have identified in 4 consanguineous families a homozygousnonsense mutation which results from a C to T change which causes theintroduction of a premature stop codon just preceding the tyrosinephosphatase domain. This same nonsense mutation was found on onechromosome of one additional family (L6) while the other chromosome hada G to A change resulting which results in a glycine to serinenon-conservative substitution. Finally, in family LD-33 an A to Ttransition results in a glutamine to leucine change in a residue locatedjust after the tyrosine phosphatase domain near the carboxy terminus.This mutation, apparently the mildest found, occurs in a family withrelative preservation of mental functions and a relatively protractedcourse (13). The five families having the C to T change are all ofSpanish decent indicating this may be the common mutation in this ethnicbackground.

[0170]FIG. 6 shows representative mutations found in 2 Lafora's diseasefamilies. The left, middle, and right panels show the in-frame sequenceof 5 codons surrounding an unaffected non-EPM2A carrier sibling, aEPM2A-carrier parent, and an affected EPM2A individual, respectively.(A) Family LD-16 in which a homozygous C to T transversion results inthe introduction of a stop mutation, and (B) Family LD-33 in which ahomozygous missense results in a glutamine to cysteine change.

[0171] The unraveling of the aetiopathogenesis of Lafora's disease needsto include an understanding of the formation of the pathognomonic Laforabodies. These unique structures have been found in LD patients in thesame tissues in which we have observed EPM2A expression (6-8, and FIG.5). Polyglucosans are unbranched equivalents of glycogen (10).Polyglucosan bodies resembling and sharing common antigenicity withLafora bodies have been found in glycogen storage disease type IV(Andersen disease) and in the normal corpora amylacea of aged brains(17). Andersen disease has been shown to arise due to mutations in thea-1,4 glucan gene on chromosome 3 which codes for the glycogen branchingenzyme (18). It is possible that mutations in a gene that lead to thelack of production of the Laforin tyrosine phosphatase protein couldaffect the metabolism of glycogen. Both glycogen biosynthesis andbreakdown are heavily regulated by phosphokinases and phosphatases (14).

[0172] EPM2A has at least two alternate forms (as does MTM1) whichappear to encode protein isoforms that might be predicted to havedifferent functions or subcellular localizations in a manner analagousto the Drosophila PTP, dPTP61F, which also undergoes alternativesplicing at the 3′ end (24,21). In the case of dPTP61F, it is known thatthe alternate carboxy termini govern the localization of the protein toeither the cytoplasmic membrane or to the nucleus (24).

[0173] Although it seems that the accumulations in Lafora bodies areresponsible for neuronal death in Lafora's disease, it is not clearwhether the epilepsy is secondary to neurodegeneration or is a directresult of abnormal neuronal Laforin expression. In various models, bothsynaptic transmission and key components of neuronal excitability suchas the NMDA type of voltage-gated calcium channels appear to be subjectto phosphoregulation (19,20).

[0174] With 75 of 500 different potential DSPs and PTPs discovered sofar, this evolving family of phosphatases is likely to have as diverseand as important a role in various regulatory processes as itscounterpart family of protein tyrosine kinases. Biological functionsattributed to these proteins so far include regulation of neuronaladhesion, control of axonal pathfinding, regulation of growth factor,cytokine and oligomeric receptor signaling, and dephosphorylation of MAPKinases (MAPKs) and other roles in tumor suppression (16). Involvementof members of this phosphatase family in non-neoplastic diseases hasbeen found in only one other human disorder, namely X-linked myotubularmyopathy (21). In this disease, mutations of the DSP MTM1 result in anarrest of muscle maturation in utero after a period of normaldevelopment (22).

[0175] Laforin is the first member of the family of PTPs and DSPs to beinvolved in human central nervous system disease. Further investigationwill be necessary to understand its role in normal brain, in theformation of Lafora bodies and in Lafora's disease and its epilepsy.

Example 3

[0176] Summary of Conunon EPM2A Mutations

[0177] Patients and Methods

[0178] Patients reported here had biopsy-proven Lafora's disease.Polymerase chain reaction (PCR) primer sequences and conditions were:JRGXBF: 5′-TCCATTGTGCTAATGCTATCTC-3′, JRGXBR:5′-TCAGCTTGCTITGAGGATATIT-3′, H1F: 5′-GAATGCTCT1TCCACTTTGC-3, PTPR:5′-GGCTCCTTAGGGAAATCAG-3′; Annealing: 620; [MgCl2]=1.25 mM. Stock DNAwas used; PCR products were purified on Qiagen columns. Restrictiondigests were performed at 370, and products were run on 3% agarose gels.

[0179] Results

[0180] Mutations

[0181] EPM2A is composed of 4 exons located within a ˜130,000 bp span ofchromosome 6q24. FIG. 11 shows a refined map of the deletion breakpointsin families LD-L4, LD9 and LD1. Filled symbols indicate patients withLD. Open rectangles on the map are the exons of EPM2A. Genomic structurearound exons 1 and 2 is shown to scale. PCR markers 365C1.H65, 266B13,D6S1703A, JRGBF/R, LDXDF/R, 109F4.E.05 and dj28H5T7 were tested. Primersequences can be obtained by looking up PAC 466P17 athttp://www.sanger.ac.uk. The positions of the forward primers of thesemarkers on the PAC are at: 58336, 59869, 98214, 108805, 123524, 124039and 132487 bp respectively. The maximum extent of the deletions areshown on the right. The deletion breakpoint regions for LD-L4 and LD9are coloured black on the map and are distinct from the deletionbreakpoint regions for LD1 are coloured grey. Each of the four deletionbreakpoints contains a MIR repeat.

[0182] As a first step towards screening exon 2 for mutations, it wasamplified by PCR with primers JRGXBF and JRGXBR. In the affected membersfrom three families, LD-L4, LD9 and LD1, no PCR product was observedindicating a possible homozygous deletion in these patients. In order toconfirm and characterize the extent of this deletion, PCR was performedwith primers covering the rest of the gene (FIG. 11). The extent of thedeletion in families LD-L4 and LD9 was determined to be ˜75,000 bpencompassing both exons 1 and 2. A smaller deletion of ˜25,000 bp wasfound in family LD1.

[0183] Screening Tests for the More Common Mutations

[0184]FIG. 12 shows restriction endonuclease screening for the twocommon mutations in exon 4. (A) Restriction map (to scale) of PCRproduct with primers H1F/PTPR. H, HaellI restriction enzyme sites one ofwhich is destroyed by the C->T mutation; boxed P, PstI site created bythe G->A mutation. (B) HaeIII and PstI digestion of the H1F/PTPR PCRproduct. Lane 1, 1 Kb ladder, lane 2: normal non-carrier individual withHaellI digestion, lanes 3 and 4: appearance of an abnormal 199 bp bandin a carrier with the C->T mutation (lane 3) and a patient with ahomozygous mutation (lane 4); lane 5: PST1 digestion does not affectnormal non-carriers, lane 6: PstI digests the PCR product into twosmaller fragments in a carrier of the G->A mutation. In patients with ahomozygous G->A mutation PSTI digestion should result in thedisappearance of the 520 bp original band. However, we presently do nothave such a patient in our data set.

[0185] The most common EPM2A mutation to date is a C->T nonsensemutation of the second base pair of exon 4 observed in 9 families (Table2). Primers H1F and PTPR amplify a 520 bp DNA fragment encompassing exon4 and including several recognition sites for the restriction enzymeHaellI, one of which is destroyed by the C->T mutation. Digestion ofthis PCR product with HaellI in normal non-carrier individuals resultsin nine small bands the largest of which is 102 bp. Digestion withHaeIII in carriers or patients results in the appearance of an abnormal199 bp band (FIGS. 12A and 12B). Carriers cannot be distinguished frompatients who carry this mutation on both chromosomes using this test(FIG. 12B).

[0186] The second most common mutation is a G→A mutation of bp 115 inexon 4 observed in 4 families (Table 2). This mutation creates a uniquePstI restriction site in the sequence of the HIF/PTPR PCR product. PstIdoes not digest this 520 bp PCR product in normal non-carrierindividuals. Carriers will therefore have one normal 520 bp band and twovariant bands of 195 bp and 315 bp (FIGS. 12A and 12B). Patientshomozygous for this mutation will only have the abnormal bands.

[0187] Finally, several families with deletions of EPM2A have beendescribed in Table 2. Two of these families (LD-L4 and LD9) appear tohave identical ˜75 Kb deletions (FIG. 11), which are different from theother two (Table 2). Nonetheless these three different deletionmutations all encompass exon 2 (FIG. 11, Table 2). Patients homozygousfor any of these deletions can be picked up by the absence of PCRamplification using primers JRGXBF/JRGXBR and appropriate controls (FIG.11).

[0188] Discussion

[0189] LD is most frequently diagnosed in societies with high rates ofconsanguinity. There also seems to be an excessive reporting fromcountries surrounding the Mediterranean basin, and many of thosefamilies appear not to be consanguineous. This initially suggested thatlike other PMEs such as Unverricht-Lundborg disease (27) or the NeuronalCeroid Lipofuscinoses (28), LD might be caused by a common mutation inmost cases. This was shown not to be the case. The large number ofdifferent mutations renders their detection for clinical purposesdifficult.

[0190] The simple DNA-based tests described above can be used to screenfor the three more common mutations in the following fashion. Digestionof the HIF/PTPR PCR product with HaeIII and PstI detects the two morecommon mutations and will confirm that an individual is a carrier of oneor the other mutation. The PstI test can further establish whether apatient or fetus is homozygous for the G->A mutation. In order toestablish if a patient is homozygous for the mutation detected by theHaeIII test, further analyses will be required such as allele specificoligonucleotide hybridization or DNA sequencing.

[0191] PCR using JRGXBF/JRGXBR will detect the deletion mutationsdescribed in this report, but only in homozygous state. This simple testcan therefore serve for prenatal or symptomatic diagnosis, but cannotdetect carriers. For carrier testing in these families further work willbe required. For example, in three of the deletions (LD-L4, LD9), thepolymorphic microsatellite marker D6S1703 is encompassed in the deletionand can be used to detect carriers by testing for loss ofheterozygosity.

[0192] The C->T mutation appears to be common in patients of Spanish (orIberian) origin (Tables 1 and 2). The −75 Kb deletion was observed intwo of two Arabic families in our data set (LD-LA and LD9).Parenthetically, LD9 is the same Arabic family described in reference 29in which two affected siblings had discordant biopsy results. Whilefalse negative biopsies are usually due to insufficient sampling and/orbiopsies done early in the course of the disease, genetic testing shouldnot have these limitations.

[0193] Additional EPM2A mutations remain to be found as presently wehave identified mutations in only 65% of families. Furthermore, we haverecently shown that an altogether different gene other than EPM2A causesLD in up to 20% of patients including the families from the FrenchCanadian province of Quebec (30). These patients are clinically andpathologically indistinguishable from those with EPM2A mutations (30).

[0194] Two deletions with different deletion breakpoints are describedin this Example. Interestingly, analysis of the sequences of thebreakpoint regions revealed the presence of the mammalian-wideinterspersed repeat (MIR) (31) in all four breakpoint regions (FIG. 11).Duplicated or repetitive sequences flanking deleted genes or exons of agene have been implicated in the generation of such deletions due tounequal recombinations. A well-studied example of this from theneurological literature is Hereditary Neuropathy with Liability toPressure Palsies. The putative mechanism in that deletion is complexinvolving a large nariner repeat which codes for a transposase thatmight facilitate the recombination (32). The role, if any, of the shortMIR repeats in the generation of the deletions in our LD patients is nowunder investigation.

[0195] In conclusion, the inventors have identified new EPM2A deletionmutations and described DNA-based screening tests for the detection ofthe more common EPM2A mutations. Further mutations in EPM2A and in theyet unidentified second gene, EPM2B, will improve the role of genetictesting and will provide insights into the function of the gene productlaforin and the pathogenesis of LD.

[0196] While the present invention has been described with reference towhat are presently considered to be the preferred examples, it is to beunderstood that the invention is not limited to the disclosed examples.To the contrary, the invention is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims.

[0197] All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

FULL CITATIONS FOR REFERENCES REFERRED TO IN THE SPECIFICATION

[0198] 1. Delgado-Escueta, A. V., Wilson, W. A., Olsen, R.O., Porter, R.J. Jasper's Basic Mechanisms of the Epilepsies (Lippincott-RavenPublishers, 1998) Chapter 1.

[0199] 2. Berkovic, S. F., Andermann, F., Carpenter, S., and Wolfe, L.S. 1986. Progressive myoclonus epilepsies: specific causes anddiagnosis. New Eng. J. Med. 315, 296-305.

[0200] 3. Minassian, B. A., Sainz, J. and Delgado-Escueta, A. V. 1996.Genetics of Myoclonic and Myoclonus epilepsies. Clin. Neuroscience3,223-235.

[0201] 4. Van Heycop Ten Ham M W. 1974. Lafora disease, a form ofprogressive myoclonus epilepsy. Handbook of Clinical Neurology15:382-422.

[0202] 5. Minassian B A, Sainz J, Bohlega S, Sakamoto L M,Delgado-Escueta A V. 1996. Genetic heterogeneity in Lafora's disease.Epilepsia 37 suppl. 5, A126.

[0203] 6. Lafora, G. R. 1911. Uber das vorkommen amyloider korperchen iminnern der ganglienzellen; zugleich ein beitrag zum studium deramyloiden substanz im nervensystem. Virchows. Arch. Path. Anat., 205,295-303.

[0204] 7. Harriman, D. G. and Millar, J. H. D. 1955. Progressivefamilial myoclonic epilepsy in 3 families: its clinical features andpathological basis. Brain 78, 325-349.

[0205] 8. Schwarz, G. A. and Yanoff, M. 1965. Lafora's disease, distinctclinico-pathologic form of Unverricht's syndrome. Arch Neurol. 12,172-188.

[0206] 9. Carpenter S and Karpati G. 1981. Sweat gland duct cells inLafora disease: Diagnosis by skin biopsy. Neurol. 31:1564-1568.

[0207] 10. Sakai, M., Austin, J., Witmer, F. and Trueb, L. 1970. Studiesin myoclonus epilepsy (Lafora body form). Neurol. 20, 160-176.

[0208] 11. Carpenter, S., Karpati, G., Andermann, F., Jacob, J. C. andAndermann, E. 1974. Lafora's disease: peroxisomal storage in skeletalmuscle. Neurol. 24, 531-538.

[0209] 12. Serratosa, J., Delgado-Escueta, A. V., Posada, I., Shih, S.,Drury, I., Berciano, J., Zabala, J. A., Antunez, M. C. and Sparkes, R.S. 1995. The gene for progressive myoclonus epilepsy of the Lafora typemaps to chromosome 6q. Hum. Molec. Genet. 9, 1657-1663.

[0210] 13. Sainz J., Minassian B. A, Serratosa J. M., Gee M. N.,Sakamoto L. M., Iranmanesh R., Bohlega S., Baumann R. J., Ryan S.,Sparkes R. S., Delgado-Escueta A. V. 1997. Lafora progressive myoclonusepilepsy: narrowing the chromosome 6q24 locus by recombinations andhomozygosities. Am. J. Hum. Genet. 61(5):1205-1209

[0211] 14. Denu J. M., Stuckey J. A., Saper M. A., Dixon J. E. 1996.Form and Function in Protein dephosphorylation. Cell 87:361-364.

[0212] 15. Yuvaniyama J., Denu J. M., Dixon J. E., Saper M. A. 1996.Crystal structure of the dual specificity protein phosphatase VHR.Science 272:1328-1331.

[0213] 16. Tonks N. K., Neel B. G. 1996. From form to function:signaling by protein tyrosine phosphatases. Cell 87:365-368.

[0214] 17. Yokota T. Ishihara T. Yoshida H. Takahashi M. Uchino F.Hamanaka S. 1988. Monoclonal antibody against polyglucosan isolated fromthe myocardium of a patient with Lafora disease. J. Neuropath. & Exp.Neurol. 47(5):572-7

[0215] 18. Thon, V. J., Khalil, M. and Cannon, J. F. 1993. Isolation ofhuman glycogen branching enzyme cDNAs be screening complementation inyeast. J. Biol. Chem. 268, 7509-7513

[0216] 19. Gurd J. W, Bissoon N. 1997. The N-methyl-D-aspartate receptorsubunits NR2A and NR2B bind to the SH2 domains of phospholipase C-gamma.J. of Neurochem. Aug;69(2):623-30

[0217] 20. Llinas R., Moreno H., Sugimori M., Mohammadi M., SchlessingerJ. 1997. Differential pre- and postsynaptic modulation of chemicaltransmission in the squid giant synapse by tyrosine phosphorylation.Proc. Nat. Acad. Sciences (USA) 94(5):1990-1994

[0218] 21. Laporte J., Hu L. J., Kretz C., Mandel J. L., Kioschis P.,Coy J. F., Klauck S. M., Poustka A., Dahl N. 1996. A gene mutated inX-linked myotubular myopathy defines a new putative tyrosine phosphatasefamily conserved in yeast. Nat. Genet. 13(2):175-82

[0219] 22. Cui X., DeVivo I., Slany R., Miyamoto A., Firestein R.,Cleary ML. 1998. Association of SET domain and myotubularin-relatedproteins modulates growth control. Nat. Genet. 18:331-337

[0220] 23. M. Kozak, 1996, Mamm. Genome 7:563.

[0221] 24. S. McLaughlin and J. E. Dixon, 1993 J. Biol. Chem., 268:6839.

[0222] 25. I. Lopes Cendes et al., 1995, Epilepsia, 36:S6.

[0223] 26. J. N. Acharya, P Satishchandra, S. K. Shankar S K. 1995,Epilepsia, 36:429.

[0224] 27. Lafreniere R G, Rochefort D L, Chretien N et al. Unstableinsertion in the 5′ flanking region of the cystatin B gene is the mostcommon mutation in progressive myoclonus epilepsy type 1, EPM1. NatGenet 1997;15:298-302

[0225] 28. Goebel H H. 7th International Congress on NeuronalCeroid-Lipofuscinoses (NCL-98), 13-16 June 1998, Dallas, USA. BrainPathol 1998;8:809-810

[0226] 29. Drury I, Blaivas M, Abou-Khalil B W, Beydoun A. Biopsyresults in a kindred with Lafora disease. Arch Neurol 1993;50:102-105

[0227] 30. Minassian B A, Sainz J, Serratosa J M et al. Genetic locusheterogeneity in Lafora's progressive myoclonus epilepsy. Ann Neurol1999;45:262-265

[0228] 31. Smit A F and Riggs A D. MIRs are classic tRNA-derived SINEsthat amplifiedbefore the mammalian radiation. Nucleic Acids Res1995;23:98-102

[0229] 32. Reiter L T, Murakami T, Koeuth T et al. A recombinationhotspot responsible for two inherited peripheral neuropathies is locatednear a mariner transposon-like element Nature Genet 1996;12:288-297TABLE 1 Summary of mutations. Mutation/ Family Genetics¹ (primers used)²Predicted effect LD-L4 consanguineous homozygous deletion deletion ofthe (D6S1703 and 109F4.E0.5) majority of EPM2A LD100-4 consanguineoushomozygous insertion of A interruption of the resulting in a frameshifttyrosine phosphatase (824F and 824R) domain I-22 consanguineoushomozygous mutation G → T glutamic acid → stop (JRGXBCF and JRGXBCFR)LD-33 consanguineous homozygous mutation A → T glutamine → leucine (824Fand 824R) LD-5 consanguineous homozygous mutation C → T arginine →cysteine (JRGXBCF and JRGXBCFR) L6 consanguineous 1. C → T (824R andH1F) 1. arginine → stop (compound 2. G → A (824F and 824R) 2. glycine →serine heterozygote) LD-16 consanguineous homozygous mutation C → Targinine → stop (824R and H1F) LD15 consanguineous homozygous mutation C→ T arginine → stop (824R and H1F) LD-48 consanguineous homozygousmutation C → T arginine → stop (824R and H1F) LD13 consanguineoushomozygous mutation C → T arginine → stop (824R and H1F) ¹Families L6,LD-16, LD15, LD-48 and LD13 are of common ethnic background. ²Thelocation of the PCR primers and mutations are shown in FIGS. 3 and 4,respectively. L Italian heterozygous mutation **arginine M G to A tostop L Non- **one mutation **arginine to stop B consanguineous codon(one Bolivian ethnicity chromosome)

[0230] TABLE 2 Most common EPM2A mutations to date Mutation n* EthnicOrigin 1 C->T nonsense 5 Spanish mutation of bp 2 of exon 4 2 1 Spanish,1 Italian 2 G->A missense mutation 1 Spanish of bp 115 of exon 4 3(a)˜75 kb deletion 2 Arabic  (b) ˜25 kb deletion 1 Iranian Total = 17

[0231] TABLE 3 Nucleotide Position Amino Acid Change Mutation (FIG. 13)(FIG. 14) C → T 721 Arg (241) → stop insert A 800 Premature stop G → A836 Gly (279) → Ser C → T 163 Glu → Stop T → G 94 Trp (32) → Gly A → G146 Asp (49) → Gly G → T 412 Glu (138) → stop A → T 878 Gln (293) → LeuDelete G 235 Premature stop G → A 179 Trp (60) → stop C → T 322 Arg(108) → Cys C → A −12 Deletion (75 kb) exons 1 and 2 Deletion (25 kb)exon 2

[0232]

1 32 1 3128 DNA Homo sapiens 1 atgcgcttcc gctttggggt ggtggtgccacccgccgtgg ccggcgcccg gccggagctg 60 ctggtggtgg ggtcgcggcc cgagctggggcgttgggagc cgcgcggtgc cgtccgcctg 120 aggccggccg gcaccgcggc gggcgacggggccctggcgc tgcaggagcc gggcctgtgg 180 ctcggggagg tggagctggc ggccgaggaggcggcgcagg acggggcgga gccgggccgc 240 gtggacacgt tctggtacaa gttcctgaagcgggagccgg gaggagagct ctcctgggaa 300 ggcaatggac ctcatcatga ccgttgctgtacttacaatg aaaacaactt ggtggatggt 360 gtgtattgtc tcccaatagg acactggattgaggccactg ggcacaccaa tgaaatgaag 420 cacacaacag acttctattt taatattgcaggccaccaag ccatgcatta ttcaagaatt 480 ctaccaaata tctggctggg tagctgccctcgtcaggtgg aacatgttac catcaaactg 540 aagcatgaat tggggattac agctgtaatgaatttccaga ctgaatggga tattgtacag 600 aattcctcag gctgtaaccg ctacccagagcccatgactc cagacactat gattaaacta 660 tatagggaag aaggcttggc ctacatctggatgccaacac cagatatgag caccgaaggc 720 cgagtacaga tgctgcccca ggcggtgtgcctgctgcatg cgctgctgga gaagggacac 780 atcgtgtacg tgcactgcaa cgctggggtgggccgctcca ccgcggctgt ctgcggctgg 840 ctccagtatg tgatgggctg gaatctgaggaaggtgcagt atttcctcat ggccaagagg 900 ccggctgtct acattgacga agaggccttggcccgggcac aagaagattt tttccagaaa 960 tttgggaagg ttcgttcttc tgtgtgtagcctgtagctgg tcagcctgct tctgccccct 1020 cctgatttcc ctaaggagcc tgggatgatgttggtcaaat gacctagaaa caaggattct 1080 acctgaactg aaaggactgt gtgacctccccaagccaacc actttcacct gggatgactt 1140 tcgattatgc tttggtttgg ggctgtatttttgaaatact ctacaagaaa gctgtggctc 1200 aacacatgag aagaagcacg aagcagttaggctgtacatc agacagaagg gtaatgcgtg 1260 cagttcctgc tgcctgcagg cagacgaggcctttgcttta cagcactgta tgtgttgcac 1320 gatggatccg tgacagcact ttcctgttgcactgaaactc ttggccatgt agaggaaaag 1380 atatggagtt atgtggattt catcactagtatgtgtgccg tgagctggtc agttgccaaa 1440 ggaggaaata aggttagaag cctgaaccgttacaaaagaa gagctcacta tggtcaaaaa 1500 gtgatggctt tcaggacttg ttttttatcctgcctcacag ttgttaaagt ctgttccaag 1560 gcatcacctt ccttctctac ccaacaaccctgtgtaacaa ctaaagtaga attatctctc 1620 atttgttggt ggtttttcct caaaattaccaaacaaagca aaaaataccc ttgtttttta 1680 tagttgagat gtcaaggaag ttaaattgaggcttaatgag cataggtagc ttgtccaagg 1740 tctcatgacc agtcaagggc aagctggagttaataatcta tatttatttg actcagcact 1800 gttttcatca caacttgttt tcccagcatcatgtagtgca tttagttttg tctttctcag 1860 ggtatagtca atatgcctgc aggagtttctatagcgagac atagaatagt attctgatca 1920 gttgccaaag aatctaggaa attagttgtattttgtgcaa gctaatttaa aaacatgatg 1980 ggctgtttta agaccagagt ggaaattcatgagaggaact atactaccaa aagagcccaa 2040 atgaccaaat ccatggataa ttgcttcacagccttggcca tcctggctca gctctcaatt 2100 tagtataata tgcagttcct gtgcctccagactatgcagc tcatcaccct aggttctaca 2160 ggaaatacag agatgaacaa ctttgccttcaaaaaatgtg ctgcctagaa aacagacctg 2220 catttcaacc caactgtaat gcaggatttggaccatgaat gatatgctag aatagaagaa 2280 agagaagtgt ttttttaatt gagagcctctatgtgcaagg tgatatataa tcatatccag 2340 tttaatcttc acaatatcca atgaagaaggtctcattatc tccatgataa agatggggaa 2400 actaaggtca gaagggttaa ctcaactgtctattgtcaca tgatgaataa atagatgaag 2460 tgagatacaa agctgggttt gattcaaagcccttactttc ctaattaaac tatgatgcgt 2520 atttattttt ctgcaccttc ctttcttccacaaacacata ttgatagatg caagagactc 2580 ttatttataa ggcgtggggg acaagaaggatacaaggtaa gtttcagtgg agctcagagg 2640 acggggagat agaactgtgg cacttaggggagatgacatt tgctttgggc agaggcagct 2700 agccaggaca catttccact ataattttacaaagttaaat ttataagcta gcattaagta 2760 aagtgaagtc cagctccctt gctaaaaataactagaggta ataattggta ttcaggtaac 2820 tcatttacag tcataatgtg ttgtgaaaatttaatcttaa aaattaaatt tttaaactat 2880 gtgggtctgt gaatttcttt aatgtctaagaaatccagct tcataatttc catgatacaa 2940 agatcttttt tcaggtggat ttttacctttgttccttttg ctctgataga caaaatcagt 3000 ttaggactat taaagaatgt tttggaataaactgtctttt tcctcaatga atgggatgtc 3060 taatgtattt caaaatcacc caaaacttttggcaaataaa agcatttaaa aagaaaaaaa 3120 aaaaaaaa 3128 2 331 PRT Homosapiens 2 Met Arg Phe Arg Phe Gly Val Val Val Pro Pro Ala Val Ala GlyAla 1 5 10 15 Arg Pro Glu Leu Leu Val Val Gly Ser Arg Pro Glu Leu GlyArg Trp 20 25 30 Glu Pro Arg Gly Ala Val Arg Leu Arg Pro Ala Gly Thr AlaAla Gly 35 40 45 Asp Gly Ala Leu Ala Leu Gln Glu Pro Gly Leu Trp Leu GlyGlu Val 50 55 60 Glu Leu Ala Ala Glu Glu Ala Ala Gln Asp Gly Ala Glu ProGly Arg 65 70 75 80 Val Asp Thr Phe Trp Tyr Lys Phe Leu Lys Arg Glu ProGly Gly Glu 85 90 95 Leu Ser Trp Glu Gly Asn Gly Pro His His Asp Arg CysCys Thr Tyr 100 105 110 Asn Glu Asn Asn Leu Val Asp Gly Val Tyr Cys LeuPro Ile Gly His 115 120 125 Trp Ile Glu Ala Thr Gly His Thr Asn Glu MetLys His Thr Thr Asp 130 135 140 Phe Tyr Phe Asn Ile Ala Gly His Gln AlaMet His Tyr Ser Arg Ile 145 150 155 160 Leu Pro Asn Ile Trp Leu Gly SerCys Pro Arg Gln Val Glu His Val 165 170 175 Thr Ile Lys Leu Lys His GluLeu Gly Ile Thr Ala Val Met Asn Phe 180 185 190 Gln Thr Glu Trp Asp IleVal Gln Asn Ser Ser Gly Cys Asn Arg Tyr 195 200 205 Pro Glu Pro Met ThrPro Asp Thr Met Ile Lys Leu Tyr Arg Glu Glu 210 215 220 Gly Leu Ala TyrIle Trp Met Pro Thr Pro Asp Met Ser Thr Glu Gly 225 230 235 240 Arg ValGln Met Leu Pro Gln Ala Val Cys Leu Leu His Ala Leu Leu 245 250 255 GluLys Gly His Ile Val Tyr Val His Cys Asn Ala Gly Val Gly Arg 260 265 270Ser Thr Ala Ala Val Cys Gly Trp Leu Gln Tyr Val Met Gly Trp Asn 275 280285 Leu Arg Lys Val Gln Tyr Phe Leu Met Ala Lys Arg Pro Ala Val Tyr 290295 300 Ile Asp Glu Glu Ala Leu Ala Arg Ala Gln Glu Asp Phe Phe Gln Lys305 310 315 320 Phe Gly Lys Val Arg Ser Ser Val Cys Ser Leu 325 330 32940 DNA Homo sapiens 3 ggtggagctg gcggccgagg aggcggcgca ggacggggcggagccgggcc gcgtggacac 60 gttctggtac aagttcctga agcgggagcc gggaggagagctctcctggg aaggcaatgg 120 acctcatcat gaccgttgct gtacttacaa tgaaaacaacttggtggatg gtgtgtattg 180 tctcccaata ggacactgga ttgaggccac tggacacaccaatgaaatga agcacacaac 240 agacttctat tttaatattg caggccacca agccatgcattattcaagaa ttctaccaaa 300 tatctggctg ggtagctgcc ctcgacaggt ggaacatgttaccatcaaac tgaagcatga 360 attggggatt acagctgtca tgaatttcca gactgaatgggatattgttc agaattcctc 420 atgctgtaac cgctacccag agcccatgac tccagacactatgattaaac tatctaggga 480 agaaggcttg gcctacatct ggatgccaac accagatatgagcaccgcag gccgagtaca 540 gatgctgccc caggcggtgt gcctgctgca tgcgctgctggagaagggac acatcgtgta 600 cgtgcactgc aacgctgggg tgggccgctc caccgcggctgtctgcggct ggctccagta 660 tgtgatgggc tggaatctga ggaaggtgca gtatttcctcatggccaaga ggccggctgt 720 ctacattgac gaagaggcct tggcccgggc acaagaagattttttccaga aatttgggaa 780 ggttcgttct tctgtgtgta gcctgtagct ggtcagcctgcttctgcccc ctcctgattt 840 ccctaaggag cctgggatga tgttggtcaa atgacctagaaacaaggatt ctacctgaac 900 tgaaaggact gtgtgacctc cccaagccaa ccactttcacctgggatgac tttcgattat 960 gctttggttt ggggctgtat ttttgaaata ctctacaagaaagctgtggc tcaacacatg 1020 agaagaagca cgaagcagtt aggctgtaca tcagacagaagggtaatgcg tgcagttcct 1080 gctgcctgca ggcagacgag gcctttgctt tacagcactgtatgtgttgc acgatggatc 1140 cgtgacagca ctttcctgtt gcactgaaac tcttggccatgtagaggaaa agatatggag 1200 ttatgtggat ttcatcacta gtatgtgtgc cgtgagctggtcagttgcca aaggaggaaa 1260 taaggttaga agcctgaacc gttacaaaag aagagctcactatggtcaaa aagtgatggc 1320 tttcaggact tgttttttat cctgcctcac agttgttaaagtctgttcca aggcatcacc 1380 ttccttctct acccaacaac cctgtgtaac aactaaagtagaattatctc tcatttgttg 1440 gtggtttttc ctcaaaatta ccaaacaaag caaaaaatacccttgttttt tatagttgag 1500 atgtcaagga agttaaattg aggcttaatg agcataggtagcttgtccaa ggtctcatga 1560 ccagtcaagg gcaagctgga gttaataatc tatatttatttgactcagca ctgttttcat 1620 cacaacttgt tttcccagca tcatgtagtg catttagttttgtctttctc agggtatagt 1680 caatatgcct gcaggagttt ctatagcgag acatagaatagtattctgat cagttgccaa 1740 agaatctagg aaattagttg tattttgtgc aagctaatttaaaaacatga tgggctgttt 1800 taagaccaga gtggaaattc atgagaggaa ctatactaccaaaagagccc aaatgaccaa 1860 atccatggat aattgcttca cagccttggc catcctggctcagctctcaa tttagtataa 1920 tatgcagttc ctgtgcctcc agactatgca gctcatcaccctaggttcta caggaaatac 1980 agagatgaac aactttgcct tcaaaaaatg tgctgcctagaaaacagacc tgcatttcaa 2040 cccaactgta atgcaggatt tggaccatga atgatatgctagaatagaag aaagagaagt 2100 gtttttttaa ttgagagcct ctatgtgcaa ggtgatatataatcatatcc agtttaatct 2160 tcacaatatc caatgaagaa ggtctcatta tctccatgataaagatgggg aaactaaggt 2220 cagaagggtt aactcaactg tctattgtca catgatgaataaatagatga agtgagatac 2280 aaagctgggt ttgattcaaa gcccttactt tcctaattaaactatgatgc gtatttattt 2340 ttctgcacct tcctttcttc cacaaacaca tattgatagatgcaagagac tcttatttat 2400 aaggcgtggg ggacaagaag gatacaaggt aagtttcagtggagctcaga ggacggggag 2460 atagaactgt ggcacttagg ggagatgaca tttgctttgggcagaggcag ctagccagga 2520 cacatttcca ctataatttt acaaagttaa atttataagctagcattaag taaagtgaag 2580 tccagctccc ttgctaaaaa taactagagg taataattggtattcaggta actcatttac 2640 agtcataatg tgttgtgaaa atttaatctt aaaaattaaatttttaaact atgtgggtct 2700 gtgaatttct ttaatgtcta agaaatccag cttcataatttccatgatac aaagatcttt 2760 tttcaggtgg atttttacct ttgttccttt tgctctgatagacaaaatca gtttaggact 2820 attaaagaat gttttggaat aaactgtctt tttcctcaatgaatgggatg tctaatgtat 2880 ttcaaaatca cccaaaactt ttggcaaata aaagcatttaaaaagaaaaa aaaaaaaaaa 2940 4 268 PRT Homo sapiens 4 Val Glu Leu Ala AlaGlu Glu Ala Ala Gln Asp Gly Ala Glu Pro Gly 1 5 10 15 Arg Val Asp ThrPhe Trp Tyr Lys Phe Leu Lys Arg Glu Pro Gly Gly 20 25 30 Glu Leu Ser TrpGlu Gly Asn Gly Pro His His Asp Arg Cys Cys Thr 35 40 45 Tyr Asn Glu AsnAsn Leu Val Asp Gly Val Tyr Cys Leu Pro Ile Gly 50 55 60 His Trp Ile GluAla Thr Gly His Thr Asn Glu Met Lys His Thr Thr 65 70 75 80 Asp Phe TyrPhe Asn Ile Ala Gly His Gln Ala Met His Tyr Ser Arg 85 90 95 Ile Leu ProAsn Ile Trp Leu Gly Ser Cys Pro Arg Gln Val Glu His 100 105 110 Val ThrIle Lys Leu Lys His Glu Leu Gly Ile Thr Ala Val Met Asn 115 120 125 PheGln Thr Glu Trp Asp Ile Val Gln Asn Ser Ser Cys Cys Asn Arg 130 135 140Tyr Pro Glu Pro Met Thr Pro Asp Thr Met Ile Lys Leu Ser Arg Glu 145 150155 160 Glu Gly Leu Ala Tyr Ile Trp Met Pro Thr Pro Asp Met Ser Thr Ala165 170 175 Gly Arg Val Gln Met Leu Pro Gln Ala Val Cys Leu Leu His AlaLeu 180 185 190 Leu Glu Lys Gly His Ile Val Tyr Val His Cys Asn Ala GlyVal Gly 195 200 205 Arg Ser Thr Ala Ala Val Cys Gly Trp Leu Gln Tyr ValMet Gly Trp 210 215 220 Asn Leu Arg Lys Val Gln Tyr Phe Leu Met Ala LysArg Pro Ala Val 225 230 235 240 Tyr Ile Asp Glu Glu Ala Leu Ala Arg AlaGln Glu Asp Phe Phe Gln 245 250 255 Lys Phe Gly Lys Val Arg Ser Ser ValCys Ser Leu 260 265 5 915 DNA Homo sapiens 5 ccaagaatcg gcacgaggattattcaagaa ttctaccaaa tatctggctg ggtagctgcc 60 ctcgacaggt ggaacatgttaccatcaaac tgaagcatga attggggatt acagctgtca 120 tgaatttcca gactgaatgggatattgttc agaattcctc atgctgtaac cgctacccag 180 agcccatgac tccagacactatgattaaac tatctaggga agaaggcttg gcctacatct 240 ggatgccaac accagatatgagcaccgcag gccgagtaca gatgctgccc caggcggtgt 300 gcctgctgca tgcgctgctggagaagggac acatcgtgta cgtgcactgc aacgctgggg 360 tgggccgctc caccgcggctgtctgcggct ggctccagta tgtgatgggc tggaatctga 420 ggaaggtgca gtatttcctcatggccaaga ggccggctgt ctacattgac gaagaggcag 480 ctagccagga cacatttccactataatttt acaaagttaa atttataagc tagcattaag 540 taaagtgaag tccagctcccttgctaaaaa taactagagg taataattgg tattcaggta 600 actcatttac agtcataatgtgttgtgaaa atttaatctt aaaaattaaa tttttaaact 660 atgtgggtct gtgaatttctttaatgtcta agaaatccag cttcataatt tccatgatac 720 aaagatcttt tttcaggtggatttttacct ttgttccttt tgctctgata gacaaaatca 780 gtttaggact attaaagaatgttttggaat aaactgtctt tttcctcaat gaatgggatg 840 tctaatgtat ttcaaaatcacccaaaactt ttggcaaata aaagcattta aaaagaaaaa 900 aaaaaaaaaa aaaaa 915 6167 PRT Homo sapiens 6 Lys Asn Arg His Glu Asp Tyr Ser Arg Ile Leu ProAsn Ile Trp Leu 1 5 10 15 Gly Ser Cys Pro Arg Gln Val Glu His Val ThrIle Lys Leu Lys His 20 25 30 Glu Leu Gly Ile Thr Ala Val Met Asn Phe GlnThr Glu Trp Asp Ile 35 40 45 Val Gln Asn Ser Ser Cys Cys Asn Arg Tyr ProGlu Pro Met Thr Pro 50 55 60 Asp Thr Met Ile Lys Leu Ser Arg Glu Glu GlyLeu Ala Tyr Ile Trp 65 70 75 80 Met Pro Thr Pro Asp Met Ser Thr Ala GlyArg Val Gln Met Leu Pro 85 90 95 Gln Ala Val Cys Leu Leu His Ala Leu LeuGlu Lys Gly His Ile Val 100 105 110 Tyr Val His Cys Asn Ala Gly Val GlyArg Ser Thr Ala Ala Val Cys 115 120 125 Gly Trp Leu Gln Tyr Val Met GlyTrp Asn Leu Arg Lys Val Gln Tyr 130 135 140 Phe Leu Met Ala Lys Arg ProAla Val Tyr Ile Asp Glu Glu Ala Ala 145 150 155 160 Ser Gln Asp Thr PhePro Leu 165 7 20 DNA Homo sapiens 7 gaatgctctt tccactttgc 20 8 19 DNAHomo sapiens 8 ggctccttag ggaaatcag 19 9 22 DNA Homo sapiens 9tccattgtgc taatgctatc tc 22 10 22 DNA Homo sapiens 10 tcagcttgctttgaggatat tt 22 11 20 DNA Homo sapiens 11 cggcacgagg attattcaag 20 1219 DNA Homo sapiens 12 gctcgggtac tgaggtctg 19 13 22 DNA Homo sapiens 13agttgttaca cagggttgtt gg 22 14 22 DNA Homo sapiens 14 aggctgtacatcagacagaa gg 22 15 22 DNA Homo sapiens 15 tccattgtgc taatgctatc tc 2216 22 DNA Homo sapiens 16 tcagcttgct ttgaggatat tt 22 17 19 DNA Homosapiens 17 gccgagtaca gatgctgcc 19 18 22 DNA Homo sapiens 18 cacacagtcctttcagttca gg 22 19 33 DNA Homo sapiens 19 gcccgggtat tcgcgccgccgccgcccgcc atg 33 20 19 DNA Homo sapiens 20 atcatgaccg ttgctgtac 19 2121 DNA Homo sapiens 21 tcatcatgac tgttgctgta c 21 22 10 PRT ArtificialSequence Description of Artificial Sequence Consensus sequence 22 HisCys Xaa Xaa Gly Xaa Xaa Arg Ser Thr 1 5 10 23 10 RNA Artificial SequenceDescription of Artificial Sequence Consensus sequence 23 cccgccaugc 1024 10 RNA Artificial Sequence Description of Artificial SequenceConsensus sequence 24 gccrccaugg 10 25 22 PRT Homo sapiens 25 Leu AlaArg Ala Gln Glu Asp Phe Phe Gln Lys Phe Gly Lys Val Arg 1 5 10 15 SerSer Val Cys Ser Leu 20 26 8 PRT Homo sapiens 26 Ala Ser Gln Asp Thr PhePro Leu 1 5 27 11 PRT Homo sapiens 27 Val His Cys Asn Ala Gly Val GlyArg Ser Thr 1 5 10 28 11 PRT Homo sapiens 28 Val His Cys Ser Asp Gly TrpAsp Arg Thr Ala 1 5 10 29 11 PRT Homo sapiens 29 Ile His Cys Lys Ala GlyLys Gly Arg Thr Gly 1 5 10 30 11 PRT Homo sapiens 30 Val His Cys Ser AlaGly Ile Gly Arg Ser Gly 1 5 10 31 11 PRT Homo sapiens 31 Val His Cys SerAla Gly Ile Gly Arg Ser Gly 1 5 10 32 11 PRT Homo sapiens 32 Val His CysGln Ala Gly Ile Ser Arg Ser Ala 1 5 10

1-19. (Cancelled)
 20. A method of detecting Lafora's disease comprisingdetecting a mutation or deletion in a nucleic acid sequence encoding aprotein tyrosine phosphatase which is associated with Lafora's diseasein a sample from an animal.
 21. A method according to claim 20comprising detecting a mutation or deletion in a region of the nucleicsequence between markers DS61003 and DS61042 as shown in FIG.
 1. 22. Amethod according to claim 20 wherein the nucleic acid sequencecomprises: (a) a nucleic acid sequence as shown in SEQ ID NO:1, SEQ IDNO:3 or SEQ ID NO:5, wherein T can also be U; (b) nucleic acid sequencescomplementary to (a); or (c) a nucleic acid molecule differing from anyof the nucleic acids of (a) or (b) in codon sequences due to thedegeneracy of the genetic code.
 23. A method according to claim 20wherein the nucleic acid sequence comprises a nucleic acid moleculehaving at least 70% identity to a nucleic acid sequence set forth in SEQID NO:1, SEQ ID NO:3 or SEQ ID NO:5.
 24. A method according to claim 20wherein the nucleic acid sequence comprises a nucleic acid moleculehaving at least 80% identity to a nucleic acid sequence set forth in SEQID NO:1, SEQ ID NO:3 or SEQ ID NO:5.
 25. A method according to claim 20wherein the nucleic acid sequence comprises a nucleic acid moleculewhich hybridizes to a nucleic acid sequence set forth in SEQ ID NO:1,SEQ ID NO:3, or SEQ ID NO:5, wherein the hybridization first occurs in asolution of 6×SSC at about 45° C., followed by a wash in a solution of2×SSC at 50° C., wherein said sequence contains a sequence encoding aprotein tyrosine phosphatase which is associated with Lafora's disease.26. A method according to claim 20 wherein the nucleic acid sequencecomprises a sequence shown in SEQ ID NO:1.
 27. A method according toclaim 20 wherein the nucleic acid sequence comprises a sequence shown inSEQ ID NO:3.
 28. A method according to claim 20 wherein the nucleic acidsequence comprises a sequence shown in SEQ ID NO:5.
 29. A methodaccording to claim 26 comprising detecting a C to T change in nucleotidenumber 721 in the sequence shown in SEQ ID NO:1.
 30. A method accordingto claim 29 wherein the C to T change is detected by a methodcomprising: (a) amplifying the nucleic acid sequences in the sample withprimers H1F (5′-GAATGCTCTTTCCACTTTGC-3) (SEQ ID NO:7) and PTPR(5′-GGCTCCTTAGGGAAATCAG-3′) (SEQ ID NO:8) in a polymerase chainreaction; (b) digesting the amplified sequences with the restrictionendonuclease HaeIII; and (c) determining the size of the digestedsequences wherein the presence of a fragment of approximately 199 bpindicates the sample is from an animal with Lafora's disease or ananimal that is a carrier of Lafora's disease.
 31. A method according toclaim 26 comprising detecting a G to A change of nucleotide number 836in the sequence shown in SEQ ID NO:1.
 32. A method according to claim 31wherein the G to A change is detected by a method comprising: (a)amplifying the nucleic acid sequences in the sample with primers H1F(5′-GAATGCTCTTTCCACTTTGC-3) (SEQ ID NO:7) and PTPR(5′-GGCTCCTTAGGGAAATCAG-3′) (SEQ ID NO:8) in a polymerase chainreaction; (b) digesting the amplified sequences with the restrictionendonuclease PstI; and (c) determining the size of the digestedsequences wherein the presence of at least one fragment of approximately520 bp indicates that the sample is from an animal that does not haveLafora's disease or an animal that is a carrier of Lafora's disease. 33.A method according to claim 26 comprising detecting a deletion of 75 kbin the sequence shown in SEQ ID NO:1.
 34. A method according to claim 26comprising detecting a deletion of 25 kb in the sequence shown in SEQ IDNO:1.
 35. A method according to claim 26, comprising detecting adeletion of about 25 kb or 75 kb in the sequence shown in SEQ ID NO:1,and further: (a) amplifying the nucleic acid sequences in the samplewith primers JRGXBF (5′-TCCATTGTGCTAATGCTATCTC-3′) (SEQ ID NO:9) andJRGXBR (5′-TCAGCTTGCTTTGAGGATATTT-3′) (SEQ ID NO:10) in a polymerasechain reaction; and (b) detecting amplified sequence wherein the absenceof an amplified sequence indicates that the sample is from an animalwith Lafora's disease.
 36. A method according to claim 26 comprisingdetecting a mutation or deletion as specified in Table 3 in a samplefrom an animal.
 37. A method according to claim 26 comprising detectingan insertion of nucleotide A at position 800 in the sequence shown inSEQ ID NO:1.
 38. A method according to claim 26 comprising detecting a Cto T change at nucleotide number 163 in the sequence shown in SEQ IDNO;
 1. 39. A method according to claim 26 comprising detecting a T to Gchange at nucleotide number 94 in the sequence shown in SEQ ID NO:1. 40.A method according to claim 26 comprising detecting a A to G change atnucleotide number 146 in the sequence shown in SEQ ID NO:1.
 41. A methodaccording to claim 26 comprising detecting a G to T change at nucleotidenumber 412 in the sequence shown in SEQ ID NO;
 1. 42. A method accordingto claim 26 comprising detecting a A to T change at nucleotide number878 in the sequence shown in SEQ ID NO;
 1. 43. A method according toclaim 26 comprising detecting a G to A change at nucleotide number 179in the sequence shown in SEQ ID NO;
 1. 44. A method according to claim26 comprising detecting a C to T change at nucleotide number 322 in thesequence shown in SEQ ID NO;
 1. 45. A method according to claim 26comprising detecting a deletion of nucleotide G at position 235 in thesequence shown in SEQ ID NO:1.
 46. A method according to claim 26comprising detecting a deletion of exons 1 and 2 in the sequence shownin SEQ ID NO:1.
 47. A method according to claim 26 comprising detectinga deletion of exon 2 in the sequence shown in SEQ ID NO:1.
 48. Anisolated protein containing a tyrosine phosphatase domain which isassociated with Lafora's disease.
 49. A protein according to claim 48having the amino acid sequence as shown in SEQ ID NO:2.
 50. A method fordetecting Lafora's disease comprising detecting a deletion or mutationin a protein according to claim
 48. 51. An isolated nucleic acidmolecule containing a sequence encoding a protein tyrosine phosphatasewhich is associated with Lafora's disease.
 52. An isolated nucleic acidmolecule according to claim 51 comprising (a) a nucleic acid sequence asshown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, wherein T can also beU; (b) nucleic acid sequences complementary to (a); (c) nucleic acidsequences which are homologous to (a) or (b); (d) a fragment of (a) to(c) that is at least 15 bases, preferably 20 to 30 bases, and which willhybridize to (a) to (d) under stringent hybridization conditions; or (e)a nucleic acid molecule differing from any of the nucleic acids of (a)to (c) in codon sequences due to the degeneracy of the genetic code. 53.An isolated nucleic acid molecule according to claim 51 having at least70% identity to a nucleic acid sequence set forth in SEQ ID NO:1, SEQ IDNO:3, or SEQ ID NO:5.
 54. The nucleic acid molecule of claim 53, whereinthe nucleic acid molecule has at least 80% identify to a nucleic acidsequence set forth in SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5.
 55. Anisolated nucleic acid molecule according to claim 51 which hybridizes toa nucleic acid sequence set forth in SEQ ID NO:1, SEQ ID NO:3, or SEQ IDNO:5, wherein the hybridization first occurs in a solution of 6×SSC atabout 45° C., followed by a wash in a solution of 2×SSC at 50° C.,wherein said sequence contains a sequence encoding a protein tyrosinephosphatase which is associated with Lafora's disease.
 56. An isolatednucleic acid molecule according to claim 51 containing a sequence whichis associated with Lafora's disease having a sequence as shown in SEQ IDNO: 1 wherein nucleotide C at position 721 is replaced with nucleotideT.
 57. An isolated nucleic acid molecule according to claim 51containing a sequence which is associated with Lafora's disease having asequence as shown in SEQ ID NO:1 wherein there is an insertion of anucleotide A at position
 800. 58. An isolated nucleic acid moleculeaccording to claim 51 containing a sequence which is associated withLafora's disease having a sequence as shown in SEQ ID NO:1 whereinnucleotide G at position 836 is replaced with nucleotide A.
 59. Anisolated nucleic acid molecule according to claim 51 containing asequence which is associated with Lafora's disease having a sequence asshown in SEQ ID NO:1 wherein nucleotide C at position 163 is replacedwith nucleotide T.
 60. An isolated nucleic acid molecule according toclaim 51 containing a sequence which is associated with Lafora's diseasehaving a sequence as shown in SEQ ID NO:1 wherein nucleotide T atposition 94 is replaced with nucleotide G.
 61. An isolated nucleic acidmolecule according to claim 51 containing a sequence which is associatedwith Lafora's disease having a sequence as shown in SEQ ID NO:1 whereinnucleotide A at position 146 is replaced with nucleotide G.
 62. Anisolated nucleic acid molecule according to claim 51 containing asequence which is associated with Lafora's disease having a sequence asshown in SEQ ID NO:1 wherein nucleotide G at position 412 is replacedwith nucleotide T.
 63. An isolated nucleic acid molecule according toclaim 51 containing a sequence which is associated with Lafora's diseasehaving a sequence as shown in SEQ ID NO:1 wherein nucleotide A atposition 878 is replaced with nucleotide T.
 64. An isolated nucleic acidmolecule according to claim 51 containing a sequence which is associatedwith Lafora's disease having a sequence as shown in SEQ ID NO:1 whereinnucleotide G at position 235 is deleted.
 65. An isolated nucleic acidmolecule according to claim 51 containing a sequence which is associatedwith Lafora's disease having a sequence as shown in SEQ ID NO:1 whereinnucleotide G at position 179 is replaced with nucleotide A.
 66. Anisolated nucleic acid molecule according to claim 51 containing asequence which is associated with Lafora's disease having a sequence asshown in SEQ ID NO:1 wherein nucleotide C at position 322 is replacedwith nucleotide T.
 67. An isolated nucleic acid molecule according toclaim 51 containing a sequence which is associated with Lafora's diseasehaving a sequence as shown in SEQ ID NO:1 wherein there is a deletion ofexons 1 and
 2. 68. An isolated nucleic acid molecule according to claim51 containing a sequence which is associated with Lafora's diseasehaving a sequence as shown in SEQ ID NO:1 wherein there is a deletion ofexon 2.